Thermostable reverse transcriptases and uses thereof

ABSTRACT

The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to reverse transcriptase enzymes which have been mutated or modified to increase thermostability, decrease terminal deoxynucleotidyl transferase activity, and/or increase fidelity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such enzymes or compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/492,194, filed Jul. 25, 2006 which is a continuation of U.S.patent application Ser. No. 10/661,819, filed Sep. 15, 2003 which claimspriority from Provisional Application No. 60/410,283, filed Sep. 13,2002. U.S. patent application Ser. No. 11/492,194 is also acontinuation-in-part of U.S. patent application Ser. No. 11/437,681,filed May 22, 2006, which is a continuation of U.S. application Ser. No.09/845,157, filed May 1, 2001, now U.S. Pat. No. 7,078,208, which claimspriority from Provisional Application No. 60/207,196, filed May 26,2000, the entire disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of molecular and cellularbiology. The invention is generally related to reverse transcriptaseenzymes and methods for the reverse transcription of nucleic acidmolecules, especially messenger RNA molecules. Specifically, theinvention relates to reverse transcriptase enzymes which have beenmutated or modified to increase thermostability, decrease terminaldeoxynucleotidyl transferase activity, and/or increase fidelity, and tomethods of producing, amplifying or sequencing nucleic acid molecules(particularly cDNA molecules) using these reverse transcriptase enzymesor compositions. The invention also relates to nucleic acid moleculesproduced by these methods and to the use of such nucleic acid moleculesto produce desired polypeptides. The invention also relates to nucleicacid molecules encoding the reverse transcriptases of the invention, tovectors containing such nucleic acid molecules, and to host cellscontaining such nucleic acid molecules. The invention also concerns kitsor compositions comprising such enzymes.

2. Related Art

cDNA and cDNA Libraries

In examining the structure and physiology of an organism, tissue orcell, it is often desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is typically manifestedupon production of the protein which the gene encodes. In order toproduce a protein, a complementary copy of one strand of the DNA doublehelix is produced by RNA polymerase enzymes, resulting in a specificsequence of ribonucleic acid (RNA). This particular type of RNA, sinceit contains the genetic message from the DNA for production of aprotein, is called messenger RNA (mRNA).

Within a given cell, tissue or organism, there exist myriad mRNAspecies, each encoding a separate and specific protein. This factprovides a powerful tool to investigators interested in studying geneticexpression in a tissue or cell. mRNA molecules may be isolated andfurther manipulated by various molecular biological techniques, therebyallowing the elucidation of the full functional genetic content of acell, tissue or organism.

One common approach to the study of gene expression is the production ofcomplementary DNA (cDNA) clones. In this technique, the mRNA moleculesfrom an organism are isolated from an extract of the cells or tissues ofthe organism. This isolation often employs solid chromatographymatrices, such as cellulose or agarose, to which oligomers of thymidine(T) have been complexed. Since the 3′ termini on most eukaryotic mRNAmolecules contain a string of adenosine (A) bases, and since A basepairs with T, the mRNA molecules can be rapidly purified from othermolecules and substances in the tissue or cell extract. From thesepurified mRNA molecules, cDNA copies may be made using the enzymereverse transcriptase (RT), which results in the production ofsingle-stranded cDNA molecules. This reaction is typically referred toas the first strand reaction. The single-stranded cDNAs may then beconverted into a complete double-stranded DNA copy (i.e., adouble-stranded cDNA) of the original mRNA (and thus of the originaldouble-stranded DNA sequence, encoding this mRNA, contained in thegenome of the organism) by the action of a DNA polymerase. Theprotein-specific double-stranded cDNAs can then be inserted into aplasmid or viral vector, which is then introduced into a host bacterial,yeast, animal or plant cell. The host cells are then grown in culturemedia, resulting in a population of host cells containing (or in manycases, expressing) the gene of interest.

This entire process, from isolation of mRNA from a source organism ortissue to insertion of the cDNA into a plasmid or vector to growth ofhost cell populations containing the isolated gene, is termed “cDNAcloning.” The set of cDNAs prepared from a given source of mRNAs iscalled a “cDNA library.” The cDNA clones in a cDNA library correspond tothe genes transcribed in the source tissue. Analysis of a cDNA librarycan yield much information on the pattern of gene expression in theorganism or tissue from which it was derived.

Retroviral Reverse Transcriptase Enzymes

Three prototypical forms of retroviral reverse transcriptase have beenstudied thoroughly. Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase contains a single subunit of 78 kDa with RNA-dependent DNApolymerase and RNase H activity. This enzyme has been cloned andexpressed in a fully active form in E. coli (reviewed in Prasad, V. R.,Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, p. 135 (1993)). Human Immunodeficiency Virus (HIV)reverse transcriptase is a heterodimer of p66 and p51 subunits in whichthe smaller subunit is derived from the larger by proteolytic cleavage.The p66 subunit has both a RNA-dependent DNA polymerase and an RNase Hdomain, while the p51 subunit has only a DNA polymerase domain. ActiveHIV p66/p51 reverse transcriptase has been cloned and expressedsuccessfully in a number of expression hosts, including E. coli(reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)).Within the HIV p66/p51 heterodimer, the 51-kD subunit is catalyticallyinactive, and the 66-kD subunit has both DNA polymerase and RNase Hactivity (Le Grice, S. F. J., et al., EMBO Journal 10:3905 (1991);Hostomsky, Z., et al., J. Viral. 66:3179 (1992)). Avian Sarcoma-LeukosisVirus (ASLV) reverse transcriptase, which includes but is not limited toRous Sarcoma Virus (RSV) reverse transcriptase, Avian MyeloblastosisVirus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV)Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis VirusMC29 Helper Virus MCAV reverse transcriptase, AvianReticuloendotheliosis Virus (REV-T) Helper Virus REV-A reversetranscriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reversetranscriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reversetranscriptase, Rous Associated Virus (RAV) reverse transcriptase, andMyeloblastosis Associated Virus (MAV) reverse transcriptase, is also aheterodimer of two subunits, α (approximately 62 kDa) and β(approximately 94 kDa), in which α is derived from β by proteolyticcleavage (reviewed in Prasad, V. R., Reverse Transcriptase, Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press (1993), p. 135). ASLVreverse transcriptase can exist in two additional catalytically activestructural forms, ββ and α (Hizi, A. and Joklik, W. K., J. Biol. Chem.252: 2281 (1977)). Sedimentation analysis suggests αβ and ββ are dimersand that the α form exists in an equilibrium between monomeric anddimeric forms (Grandgenett, D. P., et al., Proc. Nat. Acad. Sci. USA70:230 (1973); Hizi, A. and Joklik, W. K., J. Biol. Chem. 252:2281(1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA85:3372 (1988)). The ASLV αβ and ββ reverse transcriptases are the onlyknown examples of retroviral reverse transcriptase that include threedifferent activities in the same protein complex: DNA polymerase, RNaseH, and DNA endonuclease (integrase) activities (reviewed in Skalka, A.M., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1993), p. 193). The α form lacks the integrase domainand activity.

Various forms of the individual subunits of ASLV reverse transcriptasehave been cloned and expressed. These include a 98-kDa precursorpolypeptide that is normally processed proteolytically to β and a 4 kDapolypeptide removed from the β carboxy end (Alexander, F., et al., J.Virol. 61:534 (1987) and Anderson, D. et al., Focus 17:53 (1995)), andthe mature β subunit (Weis, J. H. and Salstrom, J. S., U.S. Pat. No.4,663,290 (1987); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad.Sci. USA 85:3372 (1988)). (See also Werner S, and Wohrl B. M., Eur. J.Biochem. 267:4740-4744 (2000); Werner S, and Wohrl B. M., J. Virol.74:3245-3252 (2000); Werner S, and Wohrl B. M., J. Biol. Chem.274:26329-26336 (1999).) Heterodimeric RSV αβ reverse transcriptase hasalso been purified from E. coli cells expressing a cloned RSV β gene(Chemov, A. P., et al., Biomed. Sci. 2:49 (1991)).

Reverse Transcription Efficiency

As noted above, the conversion of mRNA into cDNA by reversetranscriptase-mediated reverse transcription is an essential step in thestudy of proteins expressed from cloned genes. However, the use ofunmodified reverse transcriptase to catalyze reverse transcription isinefficient for a number of reasons. First, reverse transcriptasesometimes degrades an RNA template before the first strand reaction isinitiated or completed, primarily due to the intrinsic RNase H activitypresent in reverse transcriptase. In addition, mis-priming of the mRNAtemplate molecule can lead to the introduction of errors in the cDNAfirst strand while secondary structure of the mRNA molecule itself maymake some mRNAs refractory to first strand synthesis.

Removal of the RNase H activity of reverse transcriptase can eliminatethe first problem and improve the efficiency of reverse transcription(Gerard, G. F., et al., FOCUS 11(4):60 (1989); Gerard, G. F., et al.,FOCUS 14(3):91 (1992)). However such reverse transcriptases (“RNase H−”forms) do not address the additional problems of mis-priming and mRNAsecondary structure.

Another factor which influences the efficiency of reverse transcriptionis the ability of RNA to form secondary structures. Such secondarystructures can form, for example, when regions of RNA molecules havesufficient complementarity to hybridize and form double stranded RNA.Generally, the formation of RNA secondary structures can be reduced byraising the temperature of solutions which contain the RNA molecules.Thus, in many instances, it is desirable to reverse transcribe RNA attemperatures above 37° C. However, art known reverse transcriptasesgenerally lose activity when incubated at temperatures much above 37° C.(e.g., 50° C.).

SUMMARY OF THE INVENTION

The present invention provides, in part, reverse transcriptase enzymes,compositions comprising such enzymes and methods useful in overcominglimitations of reverse transcription discussed above. In general, theinvention provides compositions for use in reverse transcription of anucleic acid molecule, these compositions comprising one or more (e.g.,one, two, three, four, five, ten, fifteen, etc.) polypeptides having atleast one reverse transcriptase activity. Such compositions may furthercomprise one or more (e.g., one, two, three, four, five, etc.)nucleotides (e.g. one or more fluorescentl-labeled nucleotides, one ormore radiolabeled nucleotides, etc.), a suitable buffer, and/or one ormore (e.g., one, two, three, four, five, ten, fifteen, etc.) DNApolymerases. Compositions of the invention may also comprise one or more(e.g., one, two, three, four, five, ten, fifteen, etc.) oligonucleotideprimers, and/or one or more templates, and/or one or more nucleic acidmolecules (which may be complementary to all or a portion of suchtemplates).

Reverse transcriptases of the invention are preferably modified ormutated such that the thermostability of the enzyme is increased orenhanced and/or the fidelity of the enzyme is increased or enhanced. Inspecific embodiments, reverse transcriptases of the invention may besingle chained (single subunit) or multi-chained (multi-subunit) and maybe reduced or substantially reduced in RNase H activity or may have nodetectable RNase H activity or may be lacking in RNase H activity.Preferably enzymes of the invention are enzymes selected from the groupconsisting of Moloney Murine Leukemia Virus (M-MLV) RNase H− reversetranscriptase, Rous Sarcoma Virus (RSV) RNase H− reverse transcriptase,Avian Myeloblastosis Virus (AMV) RNase H− reverse transcriptase, RousAssociated Virus (RAV) RNase H− reverse transcriptase, MyeloblastosisAssociated Virus (MAV) RNase H− reverse transcriptase or other ASLVRNase H− reverse transcriptases and Human Immunodeficiency Virus (HIV)RNase H− reverse transcriptase and mutants thereof. In preferredcompositions, the reverse transcriptases are present at workingconcentrations.

In certain aspects, the invention includes reverse transcriptases whichhave been modified or mutated to increase or enhance thermostability.Examples of such reverse transcriptases include enzymes comprising oneor more modifications or mutations at positions corresponding to aminoacids selected from the group consisting of:

(a) leucine 52 of M-MLV reverse transcriptase;

(b) tyrosine 64 of M-MLV reverse transcriptase;

(c) lysine 152 of M-MLV reverse transcriptase;

(d) histidine 204 of M-MLV reverse transcriptase;

(e) methionine 289 of M-MLV reverse transcriptase;

(f) threonine 306 of M-MLV reverse transcriptase; and

(g) phenylalanine 309 of M-MLV reverse transcriptase.

In some embodiments, a modification or mutation may be the addition ofan N- and/or C-terminal tag sequence.

In specific embodiments, the invention is directed to M-MLV reversetranscriptases wherein leucine 52 is replaced with proline, tyrosine 64is replaced with arginine, lysine 152 is replaced with methionine,histidine 204 is replaced with arginine, methionine 289 is replaced withleucine, threonine 306 is replaced with either lysine or arginine,and/or phenylalanine 309 is replaced with asparagine or serine. Furtherincluded within the scope of the invention are reverse transcriptases,other than M-MLV reverse transcriptase, which contain alterationscorresponding to those set out above.

In additional aspects, the invention also include thermostable reversetranscriptases which retain at least about 50%, at least about 60%, atleast about 70%, at least about 85%, at least about 95%, at least about97%, at least about 98%, at least about 99%, at least about 100%, atleast about 150%, at least about 200%, at least about 250%, or at leastabout 300% of reverse transcriptase activity after heating to 50° C. for5 minutes.

As noted above, enzymes of the invention include reverse transcriptaseswhich exhibit reverse transcriptase activity either upon the formationof multimers (e.g., dimers) or as individual protein molecules (i.e., inmonomeric form). Examples of reverse transcriptases which exhibitreverse transcriptase activity upon the formation of multimers includeAMV, RSV and HIV reverse transcriptases. One example of a reversetranscriptase which exhibits reverse transcriptase activity as separate,individual proteins (i.e., in monomeric form) is M-MLV reversetranscriptase.

Multimeric reverse transcriptases of the invention may formhomo-multimers or hetero-multimers. In other words, the subunits of themultimeric protein complex may be identical or different. One example ofa hetero-dimeric reverse transcriptase is AMV reverse transcriptase,which is composed of two subunits that differ in primary amino acidsequence. More specifically, as already discussed, AMV reversetranscriptase may be composed of two subunits wherein one of thesesubunits is generated by proteolytic processing of the other. Thus,dimeric AMV reverse transcriptase may be composed of subunits ofdiffering size which share regions of amino acid sequence identity.

The present invention relates in particular to mutant or modifiedreverse transcriptases wherein one or more (e.g., one, two, three, four,five, ten, twelve, fifteen, twenty, etc.) amino acid changes have beenmade which renders the enzyme more thermostable in nucleic acidsynthesis, as compared to the unmutated or unmodified reversetranscriptases. Sites for mutation or modification to produce thethermostable reverse transcriptase enzymes of the present inventionand/or reverse transcriptases which exhibit other characteristics (e.g.,increased fidelity, decreased TdT activity, etc.) are listed for somereverse transcriptases in Table 1. As will be appreciated by thoseskilled in the art, one or more of the amino acids identified may bedeleted and/or replaced with one or a number of amino acid residues. Ina preferred aspect, any one or more of the amino acids identified inTable 1 may be substituted with any one or more amino acid residues suchas Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, and/or Val. The modifications described inTable 1 preferably produce thermostable reverse transcriptases of theinvention. Similar or equivalent sites or corresponding sites in otherreverse transcriptases can be mutated or modified to produce additionalthermostable reverse transcriptases, as well as reverse transcriptaseswhich exhibit other characteristics (e.g., increased fidelity, decreasedTdT activity, etc.). Thus, a reverse transcriptase of the presentinvention may have one or more of the following properties: (a)increased thermostability or increased half-life at elevatedtemperatures; (b) reduced, substantially reduced, or no detectable RNaseH activity, (c) reduced or substantially reduced terminaldeoxynucleotidyl transferase activity, and/or (d) increased fidelity. Insome embodiments, a reverse transcriptase of the invention may have aplurality of the properties listed above (e.g., a reverse transcriptasemay have enhanced thermostability, reduced RNase H activity, andenhanced fidelity).

TABLE 1 RT Amino Acids M-MLV L52, Y64, L135, H143, K152, Q165, G181,H204, I218, N249, M289, T306, F309, A517, D524, T544, V546, W548, E562,H577, D583, L604, S606, G608, F625, L626, H629, H631, H638, G641 AMV V2,L4, W12, P14, H16, T17, W20, I21, Q23, W24, L26, P27, G29, V32, Q36,L42, Q43, L44, G45, H46, I47, P49, S50, L51, S52, C53, W54, F59, I61,A64, S65, G66, S67, Y68, L70, L71, A76, A79, P83, A86, V87, Q88, Q89,G90, A91, W101, P102, L108, Q120, S131, V132, N133, N134, Q135, P137,A138, Q142, Q148, T151, Y180, M181, S190, H191, G193, A196, I201, S202,P214, V217, Q218, P221, G222, Q224, L226, G227, Y228, G231, T233, Y234,A236, P237, G239, L240, P244, I246, T248, W250, Q252, G257, Q260, W261,P264, L266, G267, L272, Y277, Q279, L280, G282, S283, P285, N286, A288,N292, L293, M297, I302, V303, L305, S306, T308, L311, L320, I332, G333,V334, G336, Q337, G338, P345, W348, L349, F350, S351, P354, A357, F358,A360, W361, L362, V364, L365, T366, T370, A374, V377, G381, C392, P400,G402, L405, G412, I414, F423, I425, A426, P428, L433, H440, P441, V443,G444, P445, A451, S453, S454, T455, H456, G458, V459, V460, W462, W468,I470, I473, A474, L476, G477, A478, S479, V480, Q481, Q482, L483, A491,W495, P496, T497, T498, P499, T500, A507, F508, M512, L513, G520, V521,P522, S523, T524, A525, A527, F528, L534, 5535, Q536, 5538, V543, 5548,H549, 5550, V552, P553, F556, T557, N560, A562 RSV V2, LA, W12, P14,H16, T17, W20, I21, Q23, W24, L26, P27, G29, V32, Q36, L42, Q43, L44,G45, H46, I47, P49, S50, L51, S52, C53, W54, F59, I61, A64, S65, G66,S67, Y68, L70, L71, A76, A79, P83, A86, V87, Q88, Q89, G90, A91, W101,P102, L108, Q120, S131, V132, N133, N134, Q135, P137, A138, Q142, Q148,T151, Y180, M181, S190, H191, G193, A196, I201, S202, P214, V217, Q218,P221, G222, Q224, L226, G227, Y228, G231, T233, Y234, A236, P237, G239,L240, P244, I246, T248, W250, Q252, G257, Q260, W261, P264, L266, G267,L272, Y277, Q279, L280, G282, S283, P285, N286, A288, N292, L293, M297,I302, V303, L305, S306, T308, L311, L320, I332, G333, V334, G336, Q337,G338, P345, W348, L349, F350, 5351, P354, A357, F358, A360, W361, L362,V364, L365, T366, T370, A374, V377, G381, C392, P400, G402, L405, G412,I414, F423, I425, A426, P428, L433, H440, P441, V443, G444, P445, A451,S453, S454, T455, H456, G458, V459, V460, W462, W468, I470, I473, A474,L476, G477, A478, S479, V480, Q481, Q482, L483, A491, W495, P496, T497,T498, P499, T500, A507, F508, M512, L513, G520, V521, P522, S523, T524,A525, A527, F528, L534, S535, Q536, S538, V543, S548, H549, S550, V552,P553, F556, T557, N560, A562 HIV Il, P3, L11, P13, G14, M15, Q22, W23,L25, T26, T38, G44, I46, S47, G50, P51, N53, P54, Y55, F60, 162, S67,T68, W70, L73, V89, Q90L91, G92, I93, 5104, V110, G111, S133, I134,N135, N136, P139, G140, I141, Q144, N146, Q150,, Y182, M183, I194, G195,Q196, T, 199, Q206, L209, P216, Q221, P224, P225, L227, M229, G230,Y231, H234, Q241, P242, V244, L245, 5250, T252, N254, Q257, G261, N264,W265, Q268, P271, G272, Q277, C279, L281, L282, G284, T285, A287, L288,T289, V291, P293, L294, T295, L300, A303, I308, L309, P312, H314, Y317,L324, I328, Q329, G332, Q333, G334, Y341, P344, F345, Y353, M356, G358,A359, H360, T361, Q372, T376, V380, Q392, W405, Q406, A407, F415, V416,N417, T418, P419, P420, L424, W425, P432, V434, G435, A436, A444, A445,N446, T449, L451, N459, G461, Q463, V465, V466, P467, L468, T469, N470,T471, T472, N473, Q474, Y482, Q486, S488, G489, L490, Q499, Y500, G503,I504, S512, S514, L516, N518, Q519, Q523, I525, W534, P536, A537, H538,G540, I541, G542, Q546, L550, S552, A553, V554, I555

Those skilled in the art will appreciate that a different isolate ofvirus may encode a reverse transcriptase enzyme having a different aminoacid at the positions identified above. Such isolates may be modified toproduce the reverse transcriptases (e.g., thermostable reversetranscriptases) of the present invention.

Reverse transcriptases of the invention may have one or more of thefollowing properties: (a) increased thermostability or increasedhalf-life at elevated temperatures; (b) reduced, substantially reduced,or no detectable RNase H activity, (c) reduced or substantially reducedterminal deoxynucleotidyl transferase activity, and/or (d) increasedfidelity.

Enzymes of the invention which have reduced or substantially reducedterminal deoxynucleotidyl transferase activity may comprise one or moremodifications or mutations at positions corresponding to amino acidsselected from the group consisting of:

(a) tyrosine 133 of M-MLV reverse transcriptase;

(b) threonine 197 of M-MLV reverse transcriptase; and

(c) phenylalanine 309 of M-MLV reverse transcriptase.

In specific embodiments, the invention is directed to M-MLV reversetranscriptases wherein tyrosine 133 is replaced with alanine, threonine197 is replaced with glutamic acid, and/or phenylalanine 309 is replacedwith asparagine. As will be appreciated, one or more of the amino acidsidentified may be deleted and/or replaced with one or a number of aminoacid residues. Further included within the scope of the invention arereverse transcriptases, other than M-MLV reverse transcriptase, whichcontain alterations corresponding to those set out above.

Additionally, enzymes which exhibit increased fidelity may comprise oneor more modifications or mutations at positions corresponding to aminoacids selected from the group consisting of:

(a) tyrosine 64 of M-MLV reverse transcriptase;

(b) arginine 116 of M-MLV reverse transcriptase;

(c) glutamine 190 of M-MLV reverse transcriptase; and

(d) valine 223 of M-MLV reverse transcriptase.

As will be appreciated, one or more of the amino acids identified may bedeleted and/or replaced with any one or a number of amino acid residues.Further, included in the invention are reverse transcriptases, otherthan M-MLV reverse transcriptase, that contain alterations correspondingto those set out above.

In some embodiments, the present invention provides a modified ormutated reverse transcriptase (e.g., preferably a modified or mutatedretroviral reverse transcriptase) having a reverse transcriptaseactivity that has a half-life of greater than that of the correspondingun-modified or un-mutated reverse transcriptase at an elevatedtemperature, i.e., greater than 37° C. In some embodiments, thehalf-life of a reverse transcriptase of the present invention may be 5minutes or greater and preferably 10 minutes or greater at 50° C. Insome embodiments, the reverse transcriptases of the invention may have ahalf-life (e.g., at 50° C.) equal to or greater than about 25 minutes,preferably equal to or greater than about 50 minutes, more preferablyequal to or greater than about 100 minutes, and most preferably, equalto or greater than about 200 minutes.

In some embodiments, the reverse transcriptases of the invention mayhave a half-life at 50° C. that is from about 10 minutes to about 200minutes, from about 10 minutes to about 150 minutes, from about 10minutes to about 100 minutes, from about 10 minutes to about 75 minutes,from about 10 minutes to about 50 minutes, from about 10 minutes toabout 40 minutes, from about 10 minutes to about 30 minutes, or fromabout 10 minutes to about 20 minutes.

A modified or mutated reverse transcriptase of the invention (e.g., onehaving a half-life at 50° C. as described above) may be a modified ormutated retroviral reverse transcriptase. A reverse transcriptaseaccording to the invention may be selected from a group consisting ofM-MLV reverse transcriptase, ASV reverse transcriptase, HIV reversetranscriptase, Avian Sarcoma-Leukosis Virus (ASLV) reversetranscriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, AvianMyeloblastosis Virus (AMV) reverse transcriptase, Avian ErythroblastosisVirus (AEV) Helper Virus MCAV reverse transcriptase, AvianMyelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase,Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reversetranscriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reversetranscriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reversetranscriptase, Rous Associated Virus (RAV) reverse transcriptase, andMyeloblastosis Associated Virus (MAV) reverse transcriptase, andfragments of any of the above having reverse transcriptase activity.

Mutated or modified reverse transcriptases of the present invention mayhave a reverse transcriptase activity (e.g., RNA-dependent DNApolymerase activity) that has a longer half-life at 55° C. than thereverse transcriptase activity of a corresponding un-mutated orun-modified reverse transcriptases. For example, introduction of theH204R, M289K, T306K, and F309N mutation into His₆-HRT increases thehalf-life at 55° C. from 1.6 minutes to 8.1 minutes (see Table 9). At55° C., the half-life of reverse transcriptase activity of a mutated ormodified reverse transcriptase of the invention may be greater thanabout 2 minutes, greater than about 3 minutes, greater than about 4minutes, greater than about 5 minutes, greater than about 6 minutes,greater than about 7 minutes, greater than about 8 minutes, greater thanabout 10 minutes, greater than about 15 minutes, greater than about 20minutes, or greater than about 30 minutes. At 55° C., the half-life ofreverse transcriptase activity of a reverse transcriptase of theinvention may be from about 2 minutes to about 60 minutes, from about 2minutes to about 45 minutes, from about 2 minutes to about 30 minutes,from about 2 minutes to about 20 minutes, from about 2 minutes to about15 minutes, from about 2 minutes to about 10 minutes, from about 2minutes to about 8 minutes, from about 2 minutes to about 7 minutes,from about 2 minutes to about 6 minutes, from about 2 minutes to about 5minutes, from about 2 minutes to about 4 minutes, or from about 2minutes to about 3 minutes. Such a reverse transcriptase may be amodified or mutant retroviral reverse transcriptase.

A modified or mutated reverse transcriptase of the invention (e.g., onehaving a half-life at 55° C. as described above) may be a modified ormutated retroviral reverse transcriptase. A mutated reversetranscriptase according to the present invention may be selected from agroup consisting of M-MLV reverse transcriptase, ASV reversetranscriptase, HIV reverse transcriptase, Avian Sarcoma-Leukosis Virus(ASLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reversetranscriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase,Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reversetranscriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAVreverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) HelperVirus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper VirusUR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAVreverse transcriptase, Rous Associated Virus (RAV) reversetranscriptase, and Myeloblastosis Associated Virus (MAV) reversetranscriptase and fragments of any of the above having reversetranscriptase activity.

Reverse transcriptases of the present invention may produce more product(e.g., full length product) at elevated temperatures than other reversetranscriptases. In one aspect, comparisons of full length productsynthesis is made at different temperatures (e.g., one temperature beinglower, such as between 37° C. and 50° C., and one temperature beinghigher, such as between 50° C. and 78° C.) while keeping all otherreaction conditions similar or the same. The amount of full lengthproduct produced may be determined using techniques well known in theart, for example, by conducting a reverse transcription reaction at afirst temperature (e.g., 37° C., 38° C., 39° C., 40° C., etc.) anddetermining the amount of full length transcript produced, conducting asecond reverse transcription reaction at a temperature higher than thefirst temperature (e.g., 45° C., 50° C., 52.5° C., 55° C., etc.) anddetermining the amount of full length product produced, and comparingthe amounts produced at the two temperatures. A convenient form ofcomparison is to determine the percentage of the amount of full lengthproduct at the first temperature that is produced at the second (i.e.,elevated) temperature. The reaction conditions used for the tworeactions (e.g., salt concentration, buffer concentration, pH, divalentmetal ion concentration, nucleoside triphosphate concentration, templateconcentration, reverse transcriptase concentration, primerconcentration, length of time the reaction is conducted, etc.) arepreferably the same for both reactions. Suitable reaction conditionsinclude, but are not limited to, a template concentration of from about1 nM to about 1 μM, from about 100 nM to 1 μM, from about 300 nM toabout 750 nM, or from about 400 nM to about 600 nM, and a reversetranscriptase concentration of from about 1 nM to about 1 μM, from about10 nM to 500 nM, from about 50 nM to about 250 nM, or from about 75 nMto about 125 nM. The ratio of the template concentration to the reversetranscriptase concentration may be from about 100:1 to about 1:1, fromabout 50:1 to about 1:1, from about 25:1 to about 1:1, from about 10:1to about 1:1, from about 5:1 to about 1:1, or from about 2.5:1 to 1:1. Areaction may be conducted from about 5 minutes to about 5 hours, fromabout 10 minutes to about 2.5 hours, from about 30 minutes to about 2hours, from about 45 minutes to about 1.5 hours, or from about 45minutes to about 1 hour. A suitable reaction time is about one hour.Other suitable reaction conditions may be determined by those skilled inthe art using routine techniques and examples of such conditions areprovided below.

When the amount of full length product produced by a reversetranscriptase of the invention at an elevated temperature is compared tothe amount of full length product produced by the same reversetranscriptase at a lower temperature, at an elevated temperature, thereverse transcriptases of the invention may produce not less than about25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 100% of the amount of fulllength product produced at the lower temperature. In some cases, thereverse transcriptases of the invention may produce an amount of fulllength product at a higher temperature that is greater than the amountof full length product produced by the reverse transcriptase at a lowertemperature (e.g., 1% to about 100% greater). In one aspect, reversetranscriptases of the invention produce approximately the same amount(e.g., no more than a 25% difference) of full length product at thelower temperature compared to the amount of full length product made atthe higher temperature.

A reverse transcriptase of the present invention may be one thatsynthesizes an amount of full length product, wherein the amount of fulllength product synthesized at 50° C. is no less than 10% (e.g., fromabout 10% to about 95%, from about 10% to about 80%, from about 10% toabout 70%, from about 10% to about 60%, from about 10% to about 50%,from about 10% to about 40%, from about 10% to about 30%, or from about10% to about 20%) of the amount of full length product it synthesizes at40° C. In some embodiments, a reverse transcriptase of the invention isone wherein the amount of full length product synthesized at 50° C. isno less than 50% (e.g., from about 50% to about 95%, from about 50% toabout 80%, from about 50% to about 70%, or from about 50% to about 60%)of the amount of full length product it synthesizes at 40° C. In someembodiments, a reverse transcriptase of the invention is one wherein theamount of full length product synthesized at 50° C. is no less than 75%(e.g., from about 75% to about 95%, from about 75%, to about 90%, fromabout 75% to about 85%, or from about 75% to about 80%) of the amount offull length product it synthesizes at 40° C. In other embodiments, areverse transcriptase of the invention is one wherein the amount of fulllength product synthesized at 50° C. is no less than 85% (e.g., fromabout 85% to about 95%, or from about 85% to about 90%) of the amount offull length product it synthesizes at 40° C.

A reverse transcriptase of the invention may be one that synthesizes anamount of full length product, wherein the amount of full length productsynthesized at 52.5° C. is no less than 10% (e.g., from about 10% toabout 30%, from about 10% to about to about 25%, from about 10% to about20%, from about 10% to about 15%, from about 20% to about 60%, fromabout 20% to about 40%, from about 20% to about 30%, from about 30% toabout 80%, from about 30% to about 60%, from about 30% to about 45%,from about 40% to about 90%, from about 40% to about 80%, from about 40%to about 60%, from about 40% to about 50% from about 50% to about 90%,or from about 50% to about 70%), of the amount of full length product itsynthesizes at 40° C. In some embodiments, the amount of full lengthproduct synthesized at 52.5° C. is no less than 30% (e.g., from about30% to about 70%, from about 30% to about 60%, from about 30% to about50%, or from about 30% to about 40%) of the amount of full lengthproduct it synthesizes at 40° C. In some embodiments, the amount of fulllength product synthesized at 52.5° C. is no less than 50% (e.g., fromabout 50% to about 70%, from about 50% to about 65%, from about 50% toabout 60%, or from about 50% to about 55%), of the amount of full lengthproduct it synthesizes at 40° C.

A reverse transcriptase of the invention may be one that synthesizes anamount of full length product, wherein the amount of full length productsynthesized at 55° C. is no less than 1% (e.g., from about 1% to about30%, from about 1% to about 25%, from about 1% to about 20%, from about1% to about 15%, from about 1% to about 10%, or from about 1% to about5%) of the amount of full length product it synthesizes at 40° C. Insome embodiments, the amount of full length product synthesized at 55°C. is no less than 5% (e.g., from about 5% to about 30%, from about 5%to about to about 25%, from about 5% to about 20%, from about 5% toabout 15%, or from about 5% to about 10%) of the amount of full lengthproduct it synthesizes at 40° C. In some embodiments, the amount of fulllength product synthesized at 55° C. is no less than 10% (e.g., fromabout 10% to about 30%, from about 10% to about to about 25%, from about10% to about 20%, from about 10% to about 15%, from about 20% to about60%, from about 20% to about 40%, from about 20% to about 30%, fromabout 30% to about 80%, from about 30% to about 60%, from about 30% toabout 45%, from about 40% to about 90%, from about 40% to about 80%,from about 40% to about 60%, from about 40% to about 50% from about 50%to about 90%, or from about 50% to about 70%) of the amount of fulllength product it synthesizes at 40° C.

In another aspect, the reverse transcriptases of the invention arecapable of producing more nucleic acid product (e.g., cDNA) and,preferably, more full length product, at one or a number of elevatedtemperatures (typically between 40° C. an 78° C.) compared to thecorresponding un-mutated or un-modified reverse transcriptase (e.g., thecontrol reverse transcriptase). Such comparisons are typically madeunder similar or the same reaction conditions and the amount of productsynthesized by the control reverse transcriptase is compared to theamount of product synthesized by the reverse transcriptase of theinvention. Preferably, the reverse transcriptases of the inventionproduce at least about 5%, at least 10%, at least 15%, at least 25%, atleast 50%, at least 75%, at least 100%, or at least 200% more product orfull length product compared to the corresponding control reversetranscriptase under the same reaction conditions and temperature. Thereverse transcriptases of the invention preferably produce from about10% to about 200%, from about 25% to about 200%, from about 50% to about200%, from about 75% to about 200%, or from about 100% to about 200%more product or full length product compared to a control reversetranscriptase under the same reaction conditions and incubationtemperature. The reverse transcriptases of the invention preferablyproduce at least 2 times, at least 3 times, at least 4 times, at least 5times, at least 6 times, at least 7 times, at least 8 times, at least 9times, at least 10 times, at least 25 times, at least 50 times, at least75 times, at least 100 times, at least 150 times, at least 200 times, atleast 300 times, at least 400 times, at least 500 times, at least 1000times, at least 5,000 times, or at least 10,000 times more product orfull length product compared to a control reverse transcriptase (e.g.,the corresponding un-mutated or un-modified reverse transcriptase) underthe same reaction conditions and temperature. The reverse transcriptasesof the invention preferably produce from 2 to 10,000, 5 to 10,000, 10 to5,000, 50 to 5,000, 50 to 500, 2 to 500, 5 to 500, 5 to 200, 5 to 100,or 5 to 75 times more product or full length product than a controlreverse transcriptase under the same reaction conditions andtemperature.

In one aspect, the reverse transcriptases of the invention produce, at50° C., at least 25% more, preferably at least 50% more and morepreferably at least 100% more nucleic acid product or full lengthproduct than a control reverse transcriptase (which is preferably thecorresponding wild-type reverse transcriptase). In another aspect, at52.5° C., the reverse transcriptases of the invention produce at least1.5 times, at least 2 times, at least 2.5 times, at least 3 times, atleast 4 times, at least 5 times, at least 6 times, at least 7 times, atleast 8 times, at least 9 times, at least 10 times the amount of nucleicacid product or full length product compared to a control reversetranscriptase. In another aspect, at 55° C., the reverse transcriptasesof the invention produce at least 2 times, at least 5 times, at least 10times, at least 15 times, at least 20 times, at least 25 times, at least50 times, at least 75 times, at least 100 times the amount of nucleicacid product or full length product compared to a control reversetranscriptase. Such comparisons are preferably made under the samereaction conditions and temperature.

Modified or mutated reverse transcriptases of the present invention mayhave an increased thermostability at elevated temperatures as comparedto corresponding un-modified or un-mutated reverse transcriptases. Theymay show increased thermostability in the presence or absence an RNAtemplate. In some instances, reverse transcriptases of the invention mayshow an increased thermostability in both the presence and absence of anRNA template. Those skilled in the art will appreciate that reversetranscriptase enzymes are typically more thermostable in the presence ofan RNA template. The increase in thermostability may be measured bycomparing suitable parameters of the modified or mutated reversetranscriptase of the invention to those of a corresponding un-modifiedor un-mutated reverse transcriptase. Suitable parameters to compareinclude, but are not limited to, the amount of product and/or fulllength product synthesized by the modified or mutated reversetranscriptase at an elevated temperature compared to the amount orproduct and/or full length product synthesized by the correspondingun-modified or un-mutated reverse transcriptase at the same temperature,and/or the half-life of reverse transcriptase activity at an elevatedtemperature of a modified or mutated reverse transcriptase at anelevated temperature compared to that of a corresponding un-modified orun-mutated reverse transcriptase.

A modified or mutated reverse transcriptase of the invention may have anincrease in thermostability at 50° C. of at least about 1.5 fold (e.g.,from about 1.5 fold to about 100 fold, from about 1.5 fold to about 50fold, from about 1.5 fold to about 25 fold, from about 1.5 fold to about10 fold) compared, for example, to the corresponding un-mutated orun-modified reverse transcriptase. A reverse transcriptase of theinvention may have an increase in thermostability at 50° C. of at leastabout 10 fold (e.g., from about 10 fold to about 100 fold, from about 10fold to about 50 fold, from about 10 fold to about 25 fold, or fromabout 10 fold to about 15 fold) compared, for example, to thecorresponding un-mutated or un-modified reverse transcriptase. A reversetranscriptase of the invention may have an increase in thermostabilityat 50° C. of at least about 25 fold (e.g., from about 25 fold to about100 fold, from about 25 fold to about 75 fold, from about 25 fold toabout 50 fold, or from about 25 fold to about 35 fold) compared to acorresponding un-mutated or un-modified reverse transcriptase.

The present invention also contemplates a modified or mutatedthermostable reverse transcriptase, wherein the reverse transcriptasehas an increase in thermostability of greater than about 1.5 fold at52.5° C. (e.g., from about 1.5 fold to about 100 fold, from about 1.5fold to about 50 fold, from about 1.5 fold to about 25 fold, or fromabout 1.5 fold to about 10 fold) compared, for example, to thecorresponding un-mutated or un-modified reverse transcriptase. A reversetranscriptase of the invention may have an increase in thermostabilityat 52.5° C. of at least about 10 fold (e.g., from about 10 fold to about100 fold, from about 10 fold to about 50 fold, from about 10 fold toabout 25 fold, or from about 10 fold to about 15 fold) compared, forexample, to the corresponding un-mutated or un-modified reversetranscriptase. A reverse transcriptase of the invention may have anincrease in thermostability at 52.5° C. of at least about 25 fold (e.g.,from about 25 fold to about 100 fold, from about 25 fold to about 75fold, from about 25 fold to about 50 fold, or from about 25 fold toabout 35 fold) compared, for example, to the corresponding un-mutated orun-modified reverse transcriptase.

In other embodiments, the present invention provides a reversetranscriptase, wherein the reverse transcriptase has an increase inthermostability of greater than about 1.5 fold at 55° C. (e.g., fromabout 1.5 fold to about 100 fold, from about 1.5 fold to about 50 fold,from about 1.5 fold to about 25 fold, or from about 1.5 fold to about 10fold) compared to a corresponding un-mutated or un-modified reversetranscriptase. In some embodiments, a reverse transcriptase of theinvention may have an increase in thermostability at 55° C. of at leastabout 10 fold (e.g., from about 10 fold to about 100 fold, from about 10fold to about 50 fold, from about 10 fold to about 25 fold, or fromabout 10 fold to about 15 fold) compared to a corresponding un-mutatedor un-modified reverse transcriptase. In some embodiments, a reversetranscriptase of the invention may have an increase in thermostabilityat 55° C. of at least about 25 fold (e.g., from about 25 fold to about100 fold, from about 25 fold to about 75 fold, from about 25 fold toabout 50 fold, or from about 25 fold to about 35 fold) compared to acorresponding un-mutated or un-modified reverse transcriptase.

The present invention provides reverse transcriptase enzymes,compositions and kits comprising such enzymes, and methods useful inpreparing labeled nucleic acid molecules by reverse transcription. Ingeneral, the invention relates to the use of polypeptides of theinvention (e.g., reverse transcriptase enzymes having one or more of themutations identified above) to synthesized labeled nucleic acidmolecules. In some embodiments, polypeptides of the invention may beheterodimers and more specifically two subunit enzymes (e.g., dimers)such as HIV RT and ASLV RTs. In some embodiments, polypeptides of theinvention may be single sub-unit enzymes (e.g., M-MLV reversetranscriptase). Preferably, such labeling involves the use of modifiednucleotides (e.g., labeled nucleotides, particularly fluorescentlylabeled nucleotides, nucleotide analogs and the like) and one or morenucleic acid templates (preferably RNA and most preferably mRNA). Inaccordance with the invention, one or more labeled nucleic acidmolecules are synthesized which are complementary to all or a portion ofthe one or more templates. The labeled nucleic acid molecules preferablyhave one or more labeled nucleotides incorporated into the synthesizedmolecule and in a preferred aspect, the labels are one or morefluorescent labels (which may be the same or different). In anotheraspect, nucleotides are used during nucleic acid synthesis using thereverse transcriptases of the invention to produce one or more nucleicacid molecules complementary to all or a portion of one or moretemplates. In such aspects, such nucleotides, which are incorporated inthe synthesized nucleic acid molecules, may be modified (before or afterincorporation) to contain one or more labels, which may then bedetected.

The invention also relates to compositions for use in the invention andsuch compositions may comprise one or more polypeptides of the invention(e.g., single sub-unit such as M-MLV RT and/or multi-subunit RTs such asHIV and ASLV RTs). Such compositions may further comprise one or morenucleotides, a suitable buffer, and/or one or more DNA polymerases. Thecompositions of the invention may also comprise one or more primers. Thereverse transcriptases in these compositions preferably have RNase Hactivity or are reduced or substantially reduced in RNase H activity,and most preferably are enzymes selected from the group consisting ofMoloney Murine Leukemia Virus (M-MLV) reverse transcriptase, RousSarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus(AMV) reverse transcriptase, Rous Associated Virus (RAV) reversetranscriptase, Myeloblastosis Associated Virus (MAV) reversetranscriptase and Human Immunodeficiency Virus (HIV) reversetranscriptase or other ASLV reverse transcriptases. The reversetranscriptases of the invention may be composed of one or more subunits(which may be the same or different). When two subunit RTs are use inthe practice of the invention, such enzymes may contain various formsand combinations of such subunits such as αβ, αα, ββ, etc. and mutants,variants or derivatives thereof. In preferred compositions, the reversetranscriptases are present at working concentrations.

The invention is also directed to methods for making one or more nucleicacid molecules and/or labeled nucleic acid molecules, comprising mixingone or more nucleic acid templates (preferably one or more RNA templatesand most preferably one or more messenger RNA templates) with one ormore polypeptides of the invention having reverse transcriptase activityand incubating the mixture under conditions sufficient to synthesize oneor more first nucleic acid molecules complementary to all or a portionof the one or more nucleic acid templates, wherein said at least one ofsaid synthesized molecules are optionally labeled and/or comprise one ormore labeled nucleotides and/or wherein said synthesized molecules mayoptionally be modified to contain one or more labels. In a preferredembodiment, the one or more first nucleic acid molecules aresingle-stranded cDNA molecules. Nucleic acid templates suitable forreverse transcription according to this aspect of the invention includeany nucleic acid molecule or population of nucleic acid molecules(preferably RNA and most preferably mRNA), particularly those derivedfrom a cell or tissue. In a preferred aspect, a population of mRNAmolecules (a number of different mRNA molecules, typically obtained fromcells or tissue) are used to make a labeled cDNA library, in accordancewith the invention. Preferred sources of nucleic acid templates includeviruses, virally infected cells, bacterial cells, fungal cells, plantcells and animal cells.

The invention also concerns methods for making one or moredouble-stranded nucleic acid molecules (which may optionally belabeled). Such methods comprise (a) mixing one or more nucleic acidtemplates (preferably RNA or mRNA, and more preferably a population ofmRNA templates) with one or more polypeptides of the invention havingreverse transcriptase activity; (b) incubating the mixture underconditions sufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of the one or more templates; and (c)incubating the one or more first nucleic acid molecules under conditionssufficient to make one or more second nucleic acid moleculescomplementary to all or a portion of the one or more first nucleic acidmolecules, thereby forming one or more double-stranded nucleic acidmolecules comprising the first and second nucleic acid molecules. Inaccordance with the invention, the first and/or second nucleic acidmolecules may be labeled (e.g., may comprise one or more of the same ordifferent labeled nucleotides and/or may be modified to contain one ormore of the same or different labels). Thus, labeled nucleotides may beused at one or both synthesis steps. Such methods may include the use ofone or more DNA polymerases as part of the process of making the one ormore double-stranded nucleic acid molecules. The invention also concernscompositions useful for making such double-stranded nucleic acidmolecules. Such compositions comprise one or more reverse transcriptasesof the invention and optionally one or more DNA polymerases, a suitablebuffer and/or one or more nucleotides (preferably including labelednucleotides).

The invention is also directed to nucleic acid molecules and/or labelednucleic acid molecules (particularly single- or double-stranded cDNAmolecules) produced according to the above-described methods and to kitscomprising these nucleic acid molecules. Such molecules or kits may beused to detect nucleic acid molecules (for example by hybridization) orfor diagnostic purposes.

The invention is also directed to kits for use in the methods of theinvention. Such kits can be used for making nucleic acid moleculesand/or labeled nucleic acid molecules (single- or double-stranded). Kitsof the invention may comprise a carrier, such as a box or carton, havingin close confinement therein one or more containers, such as vials,tubes, bottles and the like. In kits of the invention, a first containermay contain one or more of the reverse transcriptase enzymes of theinvention or one or more of the compositions of the invention. Kits ofthe invention may also comprise, in the same or different containers, atleast one component selected from one or more DNA polymerases(preferably thermostable DNA polymerases), a suitable buffer for nucleicacid synthesis and one or more nucleotides. Alternatively, thecomponents of the kit may be divided into separate containers. In oneaspect, kits of the invention comprise reverse transcriptases which haveRNase H activity or are reduced or substantially reduced in RNase Hactivity (or lacking or having undetectable RNase H activity). Such RTspreferably are selected from the group consisting of M-MLV reversetranscriptase, RSV reverse transcriptase, AMV reverse transcriptase, RAVreverse transcriptase, MAV reverse transcriptase and HIV reversetranscriptase. In additional preferred kits of the invention, theenzymes (e.g. reverse transcriptases and/or DNA polymerases) in thecontainers are present at working concentrations.

In specific embodiments, reverse transcriptases of the invention may notinclude M-MLV reverse transcriptases, HIV reverse transcriptases, AMVreverse transcriptases, and/or RSV reverse transcriptases. Thus, forexample, in certain embodiments, the invention is directed to reversetranscriptases with increased thermostability that are not a HIV reversetranscriptase. In other embodiments, the invention is directed toreverse transcriptases with increased thermostability that are not aM-MLV reverse transcriptase. In yet other embodiments, the invention isdirected to reverse transcriptases with increased thermostability thatare not an AMV reverse transcriptase. In still other embodiments, theinvention is directed to reverse transcriptases with increasedthermostability that are not a RSV reverse transcriptase.

The present invention is also directed to nucleic acid molecules (e.g.,vectors) containing a gene or nucleic acid molecules encoding the mutantor modified reverse transcriptases of the present invention (orfragments thereof including fragments having polymerase activity) and tohost cells containing such DNA or other nucleic acid molecules. Anynumber of hosts may be used to express the gene or nucleic acid moleculeof interest, including prokaryotic and eukaryotic cells. In specificembodiments, prokaryotic cells are used to express the reversetranscriptases of the invention. One example of a prokaryotic hostsuitable for use with the present invention is Escherichia coli.Examples of eukaryotic hosts suitable for use with the present inventioninclude fungal cells (e.g., Saccharomyces cerevisiae cells, Pichiapastoris cells, etc.), plant cells, and animal cells (e.g.; Drosophilamelanogaster cells, Spodoptera frugiperda Sf9 and Sf21 cells,Trichoplusa High-Five cells, C. elegans cells, Xenopus laevis cells, CHOcells, COS cells, VERO cells, BHK cells, etc.). Preferably, polypeptidesof the invention may be purified and/or isolated from a cell or organismexpressing them, which may be a wild type cell or organism or arecombinant cell or organism. In some embodiments, such polypeptides maybe substantially isolated from the cell or organism in which they areexpressed.

The invention also relates to a method of producing reversetranscriptases of the invention, said method comprising:

(a) culturing a host cell comprising a gene or other nucleic acidmolecule encoding a reverse transcriptase of the invention (preferablysuch reverse transcriptase gene or other nucleic acid molecule iscontained by a vector within the host cell);

(b) expressing the gene or nucleic acid molecule; and

(c) isolating or purifying said reverse transcriptase.

The invention is also directed to methods for making one or more (e.g.,one, two, three, four, five, ten, twelve, fifteen, etc.) nucleic acidmolecules, comprising mixing one or more (e.g., one, two, three, four,five, ten, twelve, fifteen, etc.) nucleic acid templates (preferably oneor more RNA templates and most preferably one or more messenger RNAtemplates or a population of messenger RNA templates) with one or more(e.g., one, two, three, four, five, ten, fifteen, etc.) reversetranscriptases of the invention and incubating the mixture underconditions sufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more nucleic acidtemplates. In some embodiments, the mixture is incubated at an elevatedtemperature, i.e., greater than 37° C. In specific embodiments, theelevated temperature may be from about 40° C. or greater, from about 45°C. or greater, from about 50° C. or greater, from about 51° C. orgreater, from about 52° C. or greater, from about 53° C. or greater,from about 54° C. or greater, from about 55° C. or greater, from about56° C. or greater, from about 57° C. or greater, from about 58° C. orgreater, from about 59° C. or greater, from about 60° C. or greater,from about 61° C. or greater, from about 62° C. or greater, from about63° C. or greater, from about 64° C. or greater, from about 65° C. orgreater, from about 66° C. or greater, from about 67° C. or greater,from about 68° C. or greater, from about 69° C. or greater, from about70° C. or greater, from about 71° C. or greater, from about 72° C. orgreater, from about 73° C. or greater, from about 74° C. or greater,from about 75° C. or greater, from about 76° C. or greater, from about77° C. or greater, or from about 78° C. or greater. An elevatedtemperature may be within a temperature range of from about 40° C. toabout 45° C., from about 40° C. to about 48° C., from about 40° C. toabout 50° C., from about 40° C. to about 52° C., from about 40° C. toabout 55° C., from about 40° C. to about 58° C., from about 40° C. toabout 60° C., from about 40° C. to about 65° C., from about 42° C. toabout 45° C., from about 42° C. to about 48° C., from about 42° C. toabout 50° C., from about 42° C. to about 52° C., from about 42° C. toabout 55° C., from about 42° C. to about 58° C., from about 42° C. toabout 60° C., from about 42° C. to about 65° C., from about 45° C. toabout 48° C., from about 45° C. to about 50° C., from about 45° C. toabout 52° C., from about 45° C. to about 55° C., from about 45° C. toabout 58° C., from about 45° C. to about 60° C., from about 45° C. toabout 65° C., from about 48° C. to about 50° C., from about 48° C. toabout 52° C., from about 48° C. to about 55° C., from about 48° C. toabout 58° C., from about 48° C. to about 60° C., from about 48° C. toabout 65° C., from about 50° C. to about 52° C., from about 50° C. toabout 55° C., from about 50° C. to about 58° C., from about 50° C. toabout 60° C., from about 50° C. to about 65° C., from about 52° C. toabout 55° C., from about 52° C. to about 58° C., from about 52° C. toabout 60° C., from about 52° C. to about 65° C., from about 55° C. toabout 58° C., from about 55° C. to about 60° C., from about 55° C. toabout 65° C., from about 55° C. to about 70° C., from about 58° C. toabout 60° C., from about 58° C. to about 65° C., from about 58° C. toabout 70° C. An elevated temperature may be within a temperature rangefrom about 37° C. to about 75° C., from about 40° C. to about 75° C.,from about 45° C. to about 75° C., from about 50° C. to about 75° C.,from about 51° C. to about 75° C., from about 52° C. to about 75° C.,from about 53° C. to about 75° C., from about 54° C. to about 75° C.,from about 55° C. to about 75° C. In other embodiments, the elevatedtemperature may be within the range of about 50° C. to about 70° C.,from about 51° C. to about 70° C., from about 52° C. to about 70° C.,from about 53° C. to about 70° C., from about 54° C. to about 70° C.,from about 55° C. to about 70° C., from about 56° C. to about 65° C.,from about 56° C. to about 64° C. or about 56° C. to about 62° C. Inother embodiments, the elevated temperature may be within the range ofabout 46° C. to about 60° C., from about 47° C. to about 60° C., fromabout 49° C. to about 60° C., from about 51° C. to about 60° C., fromabout 53° C. to about 60° C., or from about 54° C. to about 60° C. Inadditional specific embodiments, the first nucleic acid molecule is asingle-stranded cDNA. The invention further includes nucleic acidmolecules prepared by the above methods and reaction mixtures used inand formed by such methods. Such conditions for incubation may includethe use of one or more buffers or buffering salts, one or more primers(such as oligo dT primers) and/or one or more nucleotides (e.g.; one ormore nucleoside triphosphates). The invention also concerns compositionsfor making one or more nucleic acid molecules comprising one or morecomponents selected from the group consisting of one or more reversetranscriptases of the invention, one or more primers, one or morenucleotides and one or more suitable buffers.

Nucleic acid templates suitable for reverse transcription according tothis aspect of the invention include any nucleic acid molecule orpopulation of nucleic acid molecules (preferably RNA and most preferablymRNA), particularly those derived from a cell or tissue. In a specificaspect, a population of mRNA molecules (a number of different mRNAmolecules, typically obtained from a particular cell or tissue type) isused to make a cDNA library, in accordance with the invention. Examplesof cellular sources of nucleic acid templates include bacterial cells,fungal cells, plant cells and animal cells.

The invention also concerns methods for making one or more (e.g., one,two, three, four, five, ten, twelve, fifteen, etc.) double-strandednucleic acid molecules. Such methods comprise (a) mixing one or morenucleic acid templates (preferably RNA or mRNA, and more preferably apopulation of mRNA templates) with one or more (e.g., one, two, three,four, five, ten, fifteen, etc.) reverse transcriptases of the invention;(b) incubating the mixture under conditions sufficient to make a firstnucleic acid molecule or molecules complementary to all or a portion ofthe one or more templates; and (c) incubating the first nucleic acidmolecule or molecules under conditions sufficient to make a secondnucleic acid molecule or molecules complementary to all or a portion ofthe first nucleic acid molecule or molecules, thereby forming one ormore double-stranded nucleic acid molecules comprising the first andsecond nucleic acid molecules. In some embodiments, the incubation ofstep (b) is performed at an elevated temperature. In some embodiments,conditions may comprise the use of one or more labeled nucleotides andthe double stranded nucleic acid molecules may be labeled. In specificembodiments, the elevated temperature may be from about 40° C. orgreater, from about 45° C. or greater, from about 50° C. or greater,from about 51° C. or greater, from about 52° C. or greater, from about53° C. or greater, from about 54° C. or greater, from about 55° C. orgreater, from about 56° C. or greater, from about 57° C. or greater,from about 58° C. or greater, from about 59° C. or greater, from about60° C. or greater, from about 61° C. or greater, from about 62° C. orgreater, from about 63° C. or greater, from about 64° C. or greater,from about 65° C. or greater, from about 66° C. or greater, from about67° C. or greater, from about 68° C. or greater, from about 69° C. orgreater, from about 70° C. or greater, from about 71° C. or greater,from about 72° C. or greater, from about 73° C. or greater, from about74° C. or greater, from about 75° C. or greater, from about 76° C. orgreater, from about 77° C. or greater, or from about 78° C. or greater.An elevated temperature may be within a temperature range of from about40° C. to about 45° C., from about 40° C. to about 48° C., from about40° C. to about 50° C., from about 40° C. to about 52° C., from about40° C. to about 55° C., from about 40° C. to about 58° C., from about40° C. to about 60° C., from about 40° C. to about 65° C., from about42° C. to about 45° C., from about 42° C. to about 48° C., from about42° C. to about 50° C., from about 42° C. to about 52° C., from about42° C. to about 55° C., from about 42° C. to about 58° C., from about42° C. to about 60° C., from about 42° C. to about 65° C., from about45° C. to about 48° C., from about 45° C. to about 50° C., from about45° C. to about 52° C., from about 45° C. to about 55° C., from about45° C. to about 58° C., from about 45° C. to about 60° C., from about45° C. to about 65° C., from about 48° C. to about 50° C., from about48° C. to about 52° C., from about 48° C. to about 55° C., from about48° C. to about 58° C., from about 48° C. to about 60° C., from about48° C. to about 65° C., from about 50° C. to about 52° C., from about50° C. to about 55° C., from about 50° C. to about 58° C., from about50° C. to about 60° C., from about 50° C. to about 65° C., from about52° C. to about 55° C., from about 52° C. to about 58° C., from about52° C. to about 60° C., from about 52° C. to about 65° C., from about55° C. to about 58° C., from about 55° C. to about 60° C., from about55° C. to about 65° C., from about 55° C. to about 70° C., from about58° C. to about 60° C., from about 58° C. to about 65° C., from about58° C. to about 70° C. An elevated temperature may be within atemperature range from about 37° C. to about 75° C., from about 40° C.to about 75° C., from about 45° C. to about 75° C., from about 50° C. toabout 75° C., from about 51° C. to about 75° C., from about 52° C. toabout 75° C., from about 53° C. to about 75° C., from about 54° C. toabout 75° C., from about 55° C. to about 75° C. In other embodiments,the elevated temperature may be within the range of about 50° C. toabout 70° C., from about 51° C. to about 70° C., from about 52° C. toabout 70° C., from about 53° C. to about 70° C., from about 54° C. toabout 70° C., from about 55° C. to about 70° C., from about 56° C. toabout 65° C., from about 56° C. to about 64° C. or about 56° C. to about62° C. In other embodiments, the elevated temperature may be within therange of about 46° C. to about 60° C., from about 47° C. to about 60°C., from about 49° C. to about 60° C., from about 51° C. to about 60°C., from about 53° C. to about 60° C., or from about 54° C. to about 60°C. Such conditions may involve the use of one or more suitable buffersor buffer salts, on or more primers (such as oligo dT primers), and oneor more nucleotides. Such methods may include the use of one or more(e.g., one, two, three, four, five, ten, twelve, fifteen, etc.) DNApolymerases as part of the process of making the one or moredouble-stranded nucleic acid molecules. Such DNA polymerases arepreferably thermostable DNA polymerases and most preferably the nucleicacid synthesis accomplished with such DNA polymerases is conducted atelevated temperatures, i.e., greater than 37° C. The invention alsoconcerns compositions useful for making such double-stranded nucleicacid molecules. Such compositions comprise one or more (e.g., one, two,three, four, five, ten, twelve, fifteen, twenty, etc.) reversetranscriptases of the invention and optionally one or more DNApolymerases, a suitable buffer, one or more (e.g., one, two, three,four, five, ten, twelve, fifteen, etc.) primers, and/or one or more(e.g., one, two, three, four, five, etc.) nucleotides. The inventionfurther includes nucleic acid molecules prepared by the above methodsand reaction mixtures used in and formed by such methods.

The invention also relates to methods for amplifying a nucleic acidmolecule. Such amplification methods comprise mixing the double-strandednucleic acid molecule or molecules produced as described above with oneor more (e.g., one, two, three, four, five, ten, twelve, fifteen, etc.)DNA polymerases (preferably thermostable DNA polymerases) and incubatingthe mixture under conditions sufficient to amplify the double-strandednucleic acid molecule. In a first embodiment, the invention concerns amethod for amplifying a nucleic acid molecule, the method comprising (a)mixing one or more (e.g., one, two, three, four, five, ten, twelve,fifteen, twenty, etc.) nucleic acid templates (preferably one or moreRNA or mRNA templates and more preferably a population of mRNAtemplates) with one or more reverse transcriptases of the invention andwith one or more DNA polymerases and (b) incubating the mixture underconditions sufficient to amplify nucleic acid molecules complementary toall or a portion of the one or more templates. In some embodiments, theincubation of step (b) is performed at an elevated temperature. Inspecific embodiments, the elevated temperature may be from about 40° C.or greater, from about 45° C. or greater, from about 50° C. or greater,from about 51° C. or greater, from about 52° C. or greater, from about53° C. or greater, from about 54° C. or greater, from about 55° C. orgreater, from about 56° C. or greater, from about 57° C. or greater,from about 58° C. or greater, from about 59° C. or greater, from about60° C. or greater, from about 61° C. or greater, from about 62° C. orgreater, from about 63° C. or greater, from about 64° C. or greater,from about 65° C. or greater, from about 66° C. or greater, from about67° C. or greater, from about 68° C. or greater, from about 69° C. orgreater, from about 70° C. or greater, from about 71° C. or greater,from about 72° C. or greater, from about 73° C. or greater, from about74° C. or greater, from about 75° C. or greater, from about 76° C. orgreater, from about 77° C. or greater, or from about 78° C. or greater.An elevated temperature may be within a temperature range of from about40° C. to about 45° C., from about 40° C. to about 48° C., from about40° C. to about 50° C., from about 40° C. to about 52° C., from about40° C. to about 55° C., from about 40° C. to about 58° C., from about40° C. to about 60° C., from about 40° C. to about 65° C., from about42° C. to about 45° C., from about 42° C. to about 48° C., from about42° C. to about 50° C., from about 42° C. to about 52° C., from about42° C. to about 55° C., from about 42° C. to about 58° C., from about42° C. to about 60° C., from about 42° C. to about 65° C., from about45° C. to about 48° C., from about 45° C. to about 50° C., from about45° C. to about 52° C., from about 45° C. to about 55° C., from about45° C. to about 58° C., from about 45° C. to about 60° C., from about45° C. to about 65° C., from about 48° C. to about 50° C., from about48° C. to about 52° C., from about 48° C. to about 55° C., from about48° C. to about 58° C., from about 48° C. to about 60° C., from about48° C. to about 65° C., from about 50° C. to about 52° C., from about50° C. to about 55° C., from about 50° C. to about 58° C., from about50° C. to about 60° C., from about 50° C. to about 65° C., from about52° C. to about 55° C., from about 52° C. to about 58° C., from about52° C. to about 60° C., from about 52° C. to about 65° C., from about55° C. to about 58° C., from about 55° C. to about 60° C., from about55° C. to about 65° C., from about 55° C. to about 70° C., from about58° C. to about 60° C., from about 58° C. to about 65° C., from about58° C. to about 70° C. An elevated temperature may be within atemperature range from about 37° C. to about 75° C., from about 40° C.to about 75° C., from about 45° C. to about 75° C., from about 50° C. toabout 75° C., from about 51° C. to about 75° C., from about 52° C. toabout 75° C., from about 53° C. to about 75° C., from about 54° C. toabout 75° C., from about 55° C. to about 75° C. In other embodiments,the elevated temperature may be within the range of about 50° C. toabout 70° C., from about 51° C. to about 70° C., from about 52° C. toabout 70° C., from about 53° C. to about 70° C., from about 54° C. toabout 70° C., from about 55° C. to about 70° C., from about 56° C. toabout 65° C., from about 56° C. to about 64° C. or about 56° C. to about62° C. In other embodiments, the elevated temperature may be within therange of about 46° C. to about 60° C., from about 47° C. to about 60°C., from about 49° C. to about 60° C., from about 51° C. to about 60°C., from about 53° C. to about 60° C., or from about 54° C. to about 60°C.

Preferably, reverse transcriptases of the invention, used in methods ofthe invention, and/or present in compositions of the invention (1) arereduced or substantially reduced in RNase H activity, (2) are reduced orsubstantially reduced in TdT activity, and/or (3) exhibit increasedfidelity. Preferably, DNA polymerases used with the invention maycomprise a first DNA polymerase having 3′ exonuclease activity and asecond DNA polymerase having substantially reduced 3′ exonucleaseactivity. The invention further includes nucleic acid molecules preparedby the above methods and reaction mixtures used in and formed by suchmethods.

The invention also concerns compositions comprising one or more reversetranscriptases of the invention and one or more DNA polymerases for usein amplification reactions. Such compositions may further comprise oneor more nucleotides and/or a buffer suitable for amplification.Compositions of the invention may also comprise one or moreoligonucleotide primers. Compositions of the invention may furtherinclude nucleic acid molecules prepared by the above methods andreaction mixtures used in and formed by such methods.

The invention is also directed to nucleic acid molecules (particularlysingle- or double-stranded cDNA molecules) or amplified nucleic acidmolecules produced according to the above-described methods and tovectors (particularly expression vectors) comprising these nucleic acidmolecules or amplified nucleic acid molecules.

The invention is further directed to recombinant host cells comprisingthe above-described nucleic acid molecules, amplified nucleic acidmolecules or vectors. Examples of such host cells include bacterialcells, yeast cells, plant cells and animal cells (including insect cellsand mammalian cells).

The invention is additionally directed to methods of producingpolypeptides encoded by the nucleic acid molecules produced by themethods of the invention. Such methods include those comprisingculturing the above-described recombinant host cells and isolating theencoded polypeptides. The invention further includes polypeptidesproduced by such methods.

The invention also concerns methods for sequencing one or more (e.g.,one, two, three, four, five, ten, twelve, fifteen, etc.) nucleic acidmolecules using compositions or enzymes of the invention. Such methodscomprise (a) mixing one or more nucleic acid molecules (e.g., one ormore RNA or DNA molecules) to be sequenced with one or more reversetranscriptases of the invention, and, optionally, one or morenucleotides, one or more terminating agents, such as one or moredideoxynucleoside triphosphates, and one or more primers; (b) incubatingthe mixture under conditions sufficient to synthesize a population ofnucleic acid molecules complementary to all or a portion of the one ormore (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty,thirty, fifty, one hundred, two hundred, etc.) nucleic acid molecules tobe sequenced; and (c) separating the population of nucleic acidmolecules to determine the nucleotide sequence of all or a portion ofthe one or more nucleic acid molecules to be sequenced. Such methods mayalso comprise (a) mixing a nucleic acid molecule (e.g., one or more RNAor DNA molecules) to be sequenced with one or more primers, one or morereverse transcriptases of the invention, one or more nucleotides and oneor more terminating agents, such as one or more dideoxynucleosidetriphosphates; (b) incubating the mixture under conditions sufficient tosynthesize a population of nucleic acid molecules complementary to allor a portion of the nucleic acid molecule to be sequenced; and (c)separating members of the population of nucleic acid molecules todetermine the nucleotide sequence of all or a portion of the nucleicacid molecule to be sequenced. In some embodiments, such incubation maybe performed at elevated temperatures as described herein. The inventionfurther includes sequence data generated by the above methods, as wellas methods for generating such sequence data, and reaction mixtures usedin and formed by such methods.

The invention is also directed to kits for use in methods of theinvention. Such kits can be used for making, sequencing or amplifyingnucleic acid molecules (single- or double-stranded), preferably at theelevated temperatures described herein. Kits of the invention maycomprise a carrier, such as a box or carton, having in close confinementtherein one or more (e.g., one, two, three, four, five, ten, twelve,fifteen, etc.) containers, such as vials, tubes, bottles and the like.In kits of the invention, a first container contains one or more of thereverse transcriptase enzymes of the present invention. Kits of theinvention may also comprise, in the same or different containers, one ormore DNA polymerases (preferably thermostable DNA polymerases), one ormore (e.g., one, two, three, four, five, ten, twelve, fifteen, etc.)suitable buffers for nucleic acid synthesis, one or more nucleotides andone or more (e.g., one, two, three, four, five, ten, twelve, fifteen,etc.) oligonucleotide primers. Alternatively, the components of the kitmay be divided into separate containers (e.g., one container for eachenzyme and/or component). Kits of the invention also may compriseinstructions or protocols for carrying out the methods of the invention.In preferred kits of the invention, the reverse transcriptases arereduced or substantially reduced in RNase H activity (or lacking orhaving undetectable RNase H activity), and are most preferably selectedfrom the group consisting of M-MLV RNase H− reverse transcriptase, RSVRNase H− reverse transcriptase, AMV RNase H− reverse transcriptase, RAVRNase H− reverse transcriptase, MAV RNase H− reverse transcriptase andHIV RNase H− reverse transcriptase. In other preferred kits of theinvention, the reverse transcriptases are reduced or substantiallyreduced in TdT activity, and/or exhibit increased fidelity, as describedelsewhere herein.

In additional preferred kits of the invention, the enzymes (reversetranscriptases and/or DNA polymerases) in the containers are present atworking concentrations.

Thus, the invention is further directed to kits for use in reversetranscription, amplification or sequencing of a nucleic acid molecule,the kit comprising one or more reverse transcriptases of the invention.

As indicated above, kits of the invention may contain any number ofvarious components for practicing methods of the invention. One exampleof such a component is instructions for performing methods of theinvention. Example of such instructions include those which directindividuals using the kits to perform methods for amplifying nucleicacid molecules using one or more reverse transcriptases of theinvention.

As one skilled in the art would recognize, the full text of theseinstructions need not be included with the kit. One example of asituation in which kits of the invention would not contain such fulllength instructions is where directions are provided which informindividuals using the kits where to obtain instructions for using thekit. Thus, instructions for performing methods of the invention may beobtain from internet web pages, separately sold or distributed manualsor other product literature, etc. The invention thus includes kits whichdirect kit users to locations where they can find instructions which arenot directly packaged and/or distributed with the kits. Theseinstructions may be in any form including, but not limited to,electronic or printed forms.

The invention thus also provides, in part, kits for performing methodsusing the reverse transcriptases of the invention. In specificembodiments, kits of the invention contain instructions for performingmethods for amplifying and/or sequencing nucleic acid molecules. Thesemethods will often involve reacting RNA molecules with one or morereverse transcriptases of the invention.

In specific embodiments, reverse transcriptases of kits of the inventionmay comprise one or more modifications or mutations at positionscorresponding to amino acids selected from the group consisting of:

(a) leucine 52 of M-MLV reverse transcriptase;

(b) tyrosine 64 of M-MLV reverse transcriptase;

(c) lysine 152 of M-MLV reverse transcriptase;

(d) arginine 204 of M-MLV reverse transcriptase;

(e) methionine 289 of M-MLV reverse transcriptase;

(f) threonine 306 of M-MLV reverse transcriptase; and

(g) phenylalanine 309 of M-MLV reverse transcriptase.

Reverse transcriptases of the invention include any reversetranscriptase having one or a combination of the properties describedherein. Such properties include, but are not limited to, enhancedthermostability, reduced or eliminated RNase H activity, reducedterminal deoxynucleotidyl transferase activity, and/or increasedfidelity. Such reverse transcriptases include retroviral reversetranscriptases, bacterial reverse transcriptases, retrotransposonreverse transcriptases (e.g., reverse transcriptases of the Ty1 and/orTy3 retrotransposons), and DNA polymerases having reverse transcriptaseactivity. Preferred reverse transcriptases of the invention include asingle and multi-subunit reverse transcriptase and preferably retroviralreverse transcriptases. In particular, the invention relates toM-MLV-reverse transcriptases and ASLV-reverse transcriptases (such asAMV-RT and RSV-RT). Such reverse transcriptases of the inventionpreferably have reduced, substantially reduced, or no detectable RNase Hactivity.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of the following drawings and description of theinvention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a map of plasmid pBAD-6-His-M-MLV H− (F1).

FIG. 2A is a linear representation of the coding sequence of the M-MLVreverse transcriptase showing the locations of the restriction enzymecleavage sites used to generate the segments of the gene used togenerate mutations. FIG. 2B is a schematic representation showing theinsertion of a mutagenized PCR fragment into the coding sequence of theremaining portion of the reverse transcriptase gene.

FIG. 3 represents a scanned phosphoimage of an extension assay using (1)SUPERSCRIPT™ II reverse transcriptase, and (2) F309N. The [³²P]-labeled18-mer primer annealed to a 47-mer DNA template (5 nM) was extended byequal units of reverse transcriptase at 37° C. for 30 minutes as seen inthe extension reactions with all 4 nucleotides. The extension reactionswere analyzed by denaturing 6% gel electrophoresis. P, non-extendedprimer.

FIG. 4 represents a scanned phosphoimage showing a TdT extension assayof SUPERSCRIPT™ II reverse transcriptase and the mutants F309N, T197E,and Y133A. The [³²P]-labeled 18-mer primer annealed to a 47-mer DNAtemplate (5 nM) was extended with decreasing units of reversetranscriptase (lane (1) 646 units, lane (2) 200 units, lane (3) 50units, and lane (4) 20 units) at 37° C. for 30 minutes with all fournucleotides (see the Methods section below in Example 3). The extensionreactions were analyzed by denaturing 6% gel electrophoresis. In thisassay, extension past the 47 nucleotide templates is considerednon-template directed addition or TdT activity. P, non-extended primer.

FIG. 5 represents a scanned phosphoimage showing misinsertion assays ofSUPERSCRIPT™ II reverse transcriptase (1) and mutant protein F309Nreverse transcriptase (2) with DNA template. The [³²P]-labeled 18-merprimer annealed to a 47-mer DNA template (5 nM) was extended by equalunits of reverse transcriptase protein at 37° C. for 30 min. as seen inthe extension reactions with all four nucleotides. The extensionreactions were also performed in the presence of only 3 complementarydNTPs; minus dCTP, minus dATP, minus TTP, and minus dGTP. The extensionreactions were analyzed by denaturing 6% gel electrophoresis. In thisassay, the higher efficiency of elongation of terminated primer withonly three nucleotides will reflect the lower fidelity of theSUPERSCRIPT™ II reverse transcriptase assayed. P, non-extended primer.

FIG. 6 represents a scanned phosphoimage showing a misinsertion assay ofSUPERSCRIPT™ II reverse transcriptase (1) and mutant protein T197A/F309Nreverse transcriptase (2) and V223H/F309N (3) with DNA template. The[³²P]-labeled 18-mer primer annealed to a 47-mer DNA template (5 nM) wasextended by equal units of reverse transcriptase protein at 37° C. for30 min. as seen in the extension reactions with all four nucleotides.The extension reactions were also performed in the presence of only 3complementary dNTPs; minus dATP, and minus dCTP. The extension reactionswere analyzed by denaturing 6% gel electrophoresis. In this assay, thehigher efficiency of elongation of terminated primer with only threenucleotides will reflect the lower fidelity of the SUPERSCRIPT™ IIreverse transcriptase assayed. P, non-extended primer.

FIGS. 7A-7C show representative results obtained from the screen forthermal stable RT mutants. Lysates of mutants were assayed for RTactivity in a 96-well plate format. ³²P-Labeled DNA product was trappedon a membrane and the amount of radioactivity present was quantifiedwith a phosphorimager. FIG. 7A shows the results of an initial screen ofRT mutants in 4, 96-well plates. Heat pretreatment of lysates was at 58°C. for 10 min. RT mutants that retained the most activity after heattreatment at 58° C. were selected and lysates were screened again andthe results are shown in 7B. A duplicate screen was performed with noheat pretreatment (FIG. 7B upper panel) and heat pretreatment at 58° C.(FIG. 7B lower panel). RT mutants with the highest resistance to heatinactivation in crude extracts were purified by nickel-affinitychromatography and screened again for RT activity and the results areshown in FIG. 7C. The results after heat treatment at 37° C. are shownin FIG. 7C in the upper row, after heat treatment at 53° C. in FIG. 7Cmiddle row, and after heat treatment at 58° C. in 7C bottom row.

FIG. 8 shows a comparison of the thermal inactivation profiles of His₆H− RT and His₆ H− H204R T306K RT in crude extracts. Crude extracts weresubjected to a heat treatment in a 96-well plate for 5 min. Thetemperature of the heat treatment increased from left to right, exceptthat the wells on the far right were not heat treated.

FIG. 9 is a ribbon diagram of the crystal structure of amino acids 193to 232 of M-MLV RT showing the sites of some of the amino acidsidentified by the methods of the present invention. Potentialinteractions of arginine substituted for histidine at M-MLV RT position204 in α-helix H with E201 or T128. The catalytic site amino acids D224and D225 in the turn between β10 and β11 are also shown. Thethree-dimensional structure is taken from Georgiadis, et al., (1995)Structure 3, 879-892. Thus, the invention also includes reversetranscriptases having one or more mutations or modifications in variousregions including the α-helix H region.

FIGS. 10A and 10B are graphs of reverse transcriptase activity as afunction of Mg²⁺ concentration (FIG. 10A) and KCl concentration (FIG.10B). The DNA polymerase assay for SUPERSCRIPT™ III (SuIII) RT wasconducted at 37° C. or 50° C. for 10 minutes under variousconcentrations of A) Mg²⁺ or B) KCl. SUPERSCRIPT™ II (SuII) at 37° C.included for comparison).

FIGS. 11A and 11B show autoradiograms of TdT activity measure byextension for 60 minutes at various temperatures of a labeled DNA primeron DNA (FIG. 11A) or RNA (FIG. 11B) template forming a blunt end. T istemplate only, Lanes marked (−) is T-P plus enzyme without dNTPs. SinceSUPERSCRIPT™ III is more thermostable, its TdT activity appears greaterat 50 degrees than SUPERSCRIPT™ II.

FIGS. 12A, 12B and 12C are graphs of RT activity as a function ofincubation time. FIG. 12A shows the data obtained at 50° C., FIG. 12Bshows the data obtained at 55° C., and FIG. 12C shows the data obtainedat 60° C.

FIG. 13 is an autoradiogram comparing reverse transcriptase activity ofa variety of commercially available reverse transcriptase enzymes at 45°C., 50° C., and 55° C. SUPERSCRIPT™ III is designated SS III andSUPERSCRIPT™ II is designated SS II.

FIG. 14 is a photograph of ethidium bromide stained gels showing theresults of the evaluation of the pH of the first strand buffer. RTreactions with first-strand buffers at pHs from 8.0 to 8.8 wereperformed with 500 ng of total Hela RNA and 200 units of SUPERSCRIPT™ II(SS II) or 400 units of SUPERSCRIPT™ III (LEFN RT, which contains anN-terminal tag sequence=MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:3) and thefollowing point mutations H204R, T306K, M289L, and F309N). 2 μl of theresulting cDNA were then added to 50 μl PCR reactions containing the BF2.4 kb or Pol ε 6.8 kb primer set. Resulting PCR products were then runon a 0.8% agarose gel containing 0.4 mg/ml ethidium bromide.

FIG. 15 is a photograph of ethidium bromide stained gels showing theresults of the evaluation of the effect of temperature on the reversetranscription reaction with various reverse transcriptases. SUPERSCRIPT™II (SS II) was compared to the His-tagged EFN reverse transcriptase (Histag sequence=MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH, amino acids 1-32 of SEQID NO:2 and Table 3, EFN mutations are H204R, T306K, and F309N),His-tagged LEFN reverse transcriptase (same His tag sequence, LEFNmutations are H204R, T306K, M289L, and F309N), and to SUPERSCRIPT™ III,which is the tagged, no His LEFN reverse transcriptase (tagsequence=MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:3), LEFN mutations are H204R,T306K, M289L, and F309N).

FIG. 16 is a photograph of ethidium bromide stained gels showing theresults of the evaluation of the effect of reverse transcriptaseconcentration on the reverse transcription reaction with SUPERSCRIPT™III designated LEFN which contains the tagsequence=MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:1), and LEFN mutations, whichare H204R, T306K, M289L, and F309N.

FIG. 17 is a photograph of ethidium bromide stained gels showing theresults of the comparison of hot start RT-PCR amplification bySUPERSCRIPT™ II (Panel A) or SUPERSCRIPT™ III (Panel B). Lanes (induplicate) 1, 4, 7, and 10 are products reverse transcribed at 42° C.Lanes 2, 5, 8, and 11 are products reverse transcribed at 50° C. Lanes3, 6, 9, and 12 are products transcribed at 55° C. Lanes 1-3 are theresult of RNAs reverse transcribed by gene-specific priming from FGF,lanes 4-6 CBS 2.4, lanes 7-9 from TOP 3.2, lanes 10-12 VIN 4.6. Arrowsindicate expected product sizes of 240 bp, 2390 bp, 3162 bp, and 4641bp. SUPERSCRIPT™ III contains the tag sequence=MASGTGGQQMGRDLYDDDDKH(SEQ ID NO:3), and the LEFN mutations, which are H204R, T306K, M289L,and F309N.

FIG. 18 shows the results of RT-PCR performed with varying amountsSUPERSCRIPT™ III from 25 units to 250 units per reaction with a varietyof primer sets.

FIG. 19 shows a comparison of SUPERSCRIPT™ II (SS II) and His taggedLEFN RT in RT-PCR using 200 or 400 units in the first strand reaction.His tagged LEFN has the His tagsequence=MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH, amino acids 1-32 of SEQ IDNO:2 and Table 3, and the LEFN mutations, which are H204R, T306K, M289L,and F309N).

FIG. 20 shows the use of SUPERSCRIPT™ III (LEFN RT) in RT-PCR withvarying amounts of RT in the first strand reaction. SUPERSCRIPT™ IIIcontains the tag sequence=MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:3), and theLEFN mutations, which are H204R, T306K, M289L, and F309N.

FIG. 21 shows the results of a comparison of various primers in RT-PCRreactions using the polypeptides of the invention. EFN contains the tagsequence=MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:3), and the EFN mutations,which are H204R, T306K, and F309N.

DETAILED DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms used in recombinantDNA, virology and immunology are utilized. In order to provide a clearerand consistent understanding of the specification and claims, includingthe scope to be given such terms, the following definitions areprovided.

Cloning vector. As used herein “cloning vector” means a nucleic acidmolecule such as plasmid, cosmid, phage, phagemid or other nucleic acidmolecule which is able to replicate autonomously in a host cell, andwhich is characterized by one or a small number of recognitionsequences, (e.g., restriction endonuclease recognition sites,recombination sites, topoisomerase recognition sites, etc.) at whichsuch nucleic acid sequences may be manipulated in a determinablefashion, and into which a nucleic acid segment of interest may beinserted in order to bring about its replication and cloning. Thecloning vector may further contain a marker suitable for use in theidentification of cells transformed with the cloning vector. Markers,for example, are genes that confer a recognizable phenotype on hostcells in which such markers are expressed. Commonly used markersinclude, but are not limited to, antibiotic resistance genes such astetracycline resistance or ampicillin resistance.

Expression vector. As used herein “expression vector” means a nucleicacid molecule similar to a cloning vector but which may additionallycomprise nucleic acid sequences capable of enhancing and/or controllingthe expression of a gene or other nucleic acid molecule which has beencloned into it, after transformation into a host. The additional nucleicacid sequences may comprise promoter sequences, repressor bindingsequences and the like. The cloned gene or nucleic acid molecule isusually operably linked to one or more (e.g., one, two, three, four,etc.) of such control sequences such as promoter sequences.

Recombinant host. As used herein “recombinant” means any prokaryotic oreukaryotic or microorganism which contains the desired cloned genes ornucleic acid molecules, for example, in an expression vector, cloningvector or any nucleic acid molecule. The term “recombinant host” is alsomeant to include those host cells which have been genetically engineeredto contain the desired gene or other nucleic acid molecule on the hostchromosome or genome.

Host. As used herein “host” means any prokaryotic or eukaryotic cell ororganism that is the recipient of a replicable expression vector,cloning vector or any nucleic acid molecule. The nucleic acid moleculemay contain, but is not limited to, a structural gene, a promoter and/oran origin of replication.

Promoter. As used herein “promoter” means a nucleic acid sequencegenerally described as the 5′ region of a gene, located proximal to thestart codon which is capable of directing the transcription of a gene orother nucleic acid molecule. At the promoter region, transcription of anadjacent gene(s) or nucleic acid(s) is initiated.

Gene. As used herein “gene” means a nucleic acid sequence that containsinformation necessary for expression of a polypeptide or protein. Itincludes the promoter and the structural gene as well as other sequencesinvolved in expression of the protein.

Structural gene. As used herein “structural gene” means a DNA or othernucleic acid sequence that is transcribed into messenger RNA that isthen translated into a sequence of amino acids characteristic of aspecific polypeptide.

Operably linked. As used herein “operably linked” means that a nucleicacid element is positioned so as to influence the initiation ofexpression of the polypeptide encoded by the structural gene or othernucleic acid molecule.

Expression. As used herein “expression” refers to the process by which agene or other nucleic acid molecule produces a polypeptide. It includestranscription of the gene or nucleic acid molecule into messenger RNA(mRNA) and the translation of such mRNA into polypeptide(s).

Substantially Pure. As used herein “substantially pure” means that thedesired material is essentially free from contaminating cellularcomponents which are associated with the desired material in nature. Ina preferred aspect, a reverse transcriptase of the invention has 25% orless, preferably 15% or less, more preferably 10% or less, morepreferably 5% or less, and still more preferably 1% or lesscontaminating cellular components. In another aspect, the reversetranscriptases of the invention have no detectable protein contaminantswhen 200 units of reverse transcriptase are run on a protein gel (e.g.,SDS-PAGE) and stained with Comassie blue. Contaminating cellularcomponents may include, but are not limited to, enzymatic activitiessuch as phosphatases, exonucleases, endonucleases or undesirable DNApolymerase enzymes. Preferably, reverse transcriptases of the inventionare substantially pure.

Substantially isolated. As used herein “substantially isolated” meansthat the polypeptide of the invention is essentially free fromcontaminating proteins, which may be associated with the polypeptide ofthe invention in nature and/or in a recombinant host. In one aspect, asubstantially isolated reverse transcriptase of the invention has 25% orless, preferably 15% or less, more preferably 10% or less, morepreferably 5% or less, and still more preferably 1% or lesscontaminating proteins. In another aspect, in a sample of asubstantially isolated polypeptide of the invention, 75% or greater(preferably 80%, 85%, 90%, 95%, 98%, or 99% or greater) of the proteinin the sample is the desired reverse transcriptase of the invention. Thepercentage of contaminating protein and/or protein of interest in asample may be determined using techniques known in the art, for example,by using a protein gel (e.g., SDS-PAGE) and staining the gel with aprotein dye (e.g., Coomassie blue, silver stain, amido black, etc.). Inanother aspect, the reverse transcriptases of the invention have nodetectable protein contaminants when 200 units of reverse transcriptaseare run on a protein gel (e.g., SDS-PAGE) and stained with Comassieblue.

Primer. As used herein “primer” refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a DNA molecule.

Template. The term “template” as used herein refers to a double-strandedor single-stranded nucleic acid molecule which is to be amplified,copied or sequenced. In the case of a double-stranded DNA molecule,denaturation of its strands to form single-stranded first and secondstrands may be performed before these molecules are amplified, copied orsequenced. A primer complementary to a portion of a nucleic acidtemplate is hybridized under appropriate conditions and a nucleic acidpolymerase, such as the reverse transcriptase enzymes of the invention,may then add nucleotide monomers to the primer thereby synthesizing anucleic acid molecule complementary to said template or a portionthereof. The newly synthesized nucleic acid molecule, according to theinvention, may be equal or shorter in length than the original template.Mismatch incorporation during the synthesis or extension of the newlysynthesized nucleic acid molecule may result in one or a number ofmismatched base pairs. Thus, the synthesized nucleic acid molecule neednot be exactly complementary to the template.

Incorporating. The term “incorporating” as used herein means becoming apart of a nucleic acid molecule or primer.

Oligonucleotide. “Oligonucleotide” refers to a synthetic or naturalmolecule comprising a covalently linked sequence of nucleotides whichare joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

Nucleotide. As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, andnucleotide derivatives that confer nuclease resistance on the nucleicacid molecule containing them. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (ddNTPs) and theirderivatives. Illustrated examples of dideoxyribonucleoside triphosphatesinclude, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.According to the present invention, a “nucleotide” may be unlabeled ordetectably labeled by well known techniques. Detectable labels include,for example, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels. Fluorescent labels ofnucleotides may include but are not limited fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′ dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanineand 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specificexamples of fluorescently labeled nucleotides include [R6G]dUTP,[TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP,[FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP,[dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from PerkinElmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLinkCy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLinkCy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham ArlingtonHeights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP,Tetramethyl-rodamine-6-dUTP, IR₇₇₀-9-dATP, Fluorescein-12-ddUTP,Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from BoehringerMannheim Indianapolis, Ind.; and ChromaTide Labeled Nucleotides,BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP,BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, CascadeBlue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP,fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP,Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP,tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, andTexas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.

Probes. The term probes refer to single or double stranded nucleic acidmolecules or oligonucleotides which are detectably labeled by one ormore detectable markers or labels. Such labels or markers may be thesame or different and may include radioactive labels, fluorescentlabels, chemiluminescent labels, bioluminescent labels and enzymelabels, although one or more fluorescent labels (which are the same ordifferent) are preferred in accordance with the invention. Probes havespecific utility in the detection of nucleic acid molecules byhybridization and thus may be used in diagnostic assays.

Hybridization. As used herein, hybridization (hybridizing) refers to thepairing of two complementary single-stranded nucleic acid molecules (RNAand/or DNA) to give a double-stranded molecule. As one skilled in theart will recognize, two nucleic acid molecules may be hybridized,although the base pairing is not completely complementary. Accordingly,mismatched bases do not prevent hybridization of two nucleic acidmolecules provided that appropriate conditions, well known in the art,are used.

Thermostable Reverse Transcriptase. For the purposes of this disclosure,a thermostable reverse transcriptase includes a reverse transcriptasewhich retains a greater percentage or amount of its activity after aheat treatment than is retained by a reverse transcriptase that haswild-type thermostability after an identical treatment. Thus, a reversetranscriptase having increased/enhanced thermostability may be definedas a reverse transcriptase having any increase in thermostability,preferably from about 1.2 to about 10,000 fold, from about 1.5 to about10,000 fold, from about 2 to about 5,000 fold, or from about 2 to about2000 fold (preferably greater than about 5 fold, more preferably greaterthan about 10 fold, still more preferably greater than about 50 fold,still more preferably greater than about 100 fold, still more preferablygreater than about 500 fold, and most preferably greater than about 1000fold) retention of activity after a heat treatment sufficient to cause areduction in the activity of a reverse transcriptase that is wild-typefor thermostability. Preferably, the mutant or modified reversetranscriptase of the invention is compared to the correspondingunmodified or wild-type reverse transcriptase to determine the relativeenhancement or increase in thermostability. For example, after a heattreatment at 52° C. for 5 minutes, a thermostable reverse transcriptasemay retain approximately 90% of the activity present before the heattreatment, whereas a reverse transcriptase that is wild-type forthermostability may retain 10% of its original activity. Likewise, aftera heat treatment at 53° C. for five minutes, a thermostable reversetranscriptase may retain approximately 80% of its original activity,whereas a reverse transcriptase that is wild-type for thermostabilitymay have no measurable activity. Similarly, after a heat treatment at50° C. for five minutes, a thermostable reverse transcriptase may retainapproximately 50%, approximately 55%, approximately 60%, approximately65%, approximately 70%, approximately 75%, approximately 80%,approximately 85%, approximately 90%, or approximately 95% of itsoriginal activity, whereas a reverse transcriptase that is wild-type forthermostability may have no measurable activity or may retain 10%, 15%or 20% of its original activity. In the first instance (i.e., after heattreatment at 52° C. for 5 minutes), the thermostable reversetranscriptase would be said to be 9-fold more thermostable than thewild-type reverse transcriptase. Examples of conditions which may beused to measure thermostability of reverse transcriptases are set outbelow, for example, in the Examples.

The thermostability of a reverse transcriptase can be determined bycomparing the residual activity of a sample of the reverse transcriptasethat has been subjected to a heat treatment, i.e., incubated at 52° C.for a given period of time, for example, five minutes, to a controlsample of the same reverse transcriptase that has been incubated at roomtemperature for the same length of time as the heat treatment. Typicallythe residual activity may be measured by following the incorporation ofa radiolabled deoxyribonucleotide into an oligodeoxyribonucleotideprimer using a complementary oligoribonucleotide template. For example,the ability of the reverse transcriptase to incorporate [α-³²P]-dGTPinto an oligo-dG primer using a poly(riboC) template may be assayed todetermine the residual activity of the reverse transcriptase.

In another aspect, thermostable reverse transcriptases of the inventionmay include any reverse transcriptase which is inactivated at a highertemperature compared to the corresponding wild-type, unmutated, orunmodified reverse transcriptase. Preferably, the inactivationtemperature for the thermostable reverse transcriptases of the inventionis from about 2° C. to about 50° C. (e.g., about 2° C., about 4° C.,about 5° C., about 8° C., about 10° C., about 12° C., about 14° C.,about 16° C., about 18° C., about 20° C., about 24° C., about 26° C.,about 28° C., about 30° C., about 33° C., about 35° C., about 38° C.,about 40° C., about 42° C., about 44° C., about 46° C., about 48° C., orabout 50° C.) higher than the inactivation temperature for thecorresponding wild-type, unmutated, or unmodified reverse transcriptase.More preferably, the inactivation temperature for the reversetranscriptases of the invention is from about 5° C. to about 50° C.,from about 5° C. to about 40° C., from about 5° C. to about 30° C., orfrom about 5° C. to about 25° C. greater than the inactivationtemperature for the corresponding wild-type, unmutated or unmodifiedreverse transcriptase, when compared under the same conditions.

The difference in inactivation temperature for the reverse transcriptaseof the invention compared to its corresponding wild-type, unmutated orunmodified reverse transcriptase can be determined by treating samplesof such reverse transcriptases at different temperatures for a definedtime period and then measuring residual reverse transcriptase activity,if any, after the samples have been heat treated. Determination of thedifference or delta in the inactivation temperature between the testreverse transcriptase compared to the wild-type, unmutated or unmodifiedcontrol is determined by comparing the difference in temperature atwhich each reverse transcriptase is inactivated (i.e., no residualreverse transcriptase activity is measurable in the particular assayused). As will be recognized, any number of reverse transcriptase assaysmay be used to determine the different or delta of inactivationtemperatures for any reverse transcriptases tested.

In another aspect, thermostability of a reverse transcriptase of theinvention may be determined by measuring the half-life of the reversetranscriptase activity of a reverse transcriptase of interest. Suchhalf-life may be compared to a control or wild-type reversetranscriptase to determine the difference (or delta) in half-life.Half-lifes of the reverse transcriptases of the invention are preferablydetermined at elevated temperatures (e.g., greater than 37° C.) andpreferably at temperatures ranging from 40° C. to 80° C., morepreferably at temperatures ranging from 45° C. to 75° C., 50° C. to 70°C., 50° C. to 65° C., and 50° C. to 60° C. Preferred half-lifer of thereverse transcriptases of the invention may range from 4 minutes to 10hours, 4 minutes to 7.5 hours, 4 minutes to 5 hours, 4 minutes to 2.5hours, or 4 minutes to 2 hours, depending upon the temperature used. Forexample, the reverse transcriptase activity of the reversetranscriptases of the invention may have a half-life of at least 4minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, atleast 8 minutes, at least 9 minutes, at least 10 minutes, at least 11minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes,at least 15 minutes, at least 20 minute, at least 25 minutes, at least30 minutes, at least 40 minutes, at least 50 minutes, at least 60minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes,at least 100 minutes, at least 115 minutes, at least 125 minutes, atleast 150 minutes, at least 175 minutes, at least 200 minutes, at least225 minutes, at least 250 minutes, at least 275 minutes, at least 300minutes, at least 400 minutes, at least 500 minutes at temperatures of48° C., 50° C., 52° C., 52.5° C., 55° C., 57° C., 60° C., 62° C., 65°C., 68° C., and/or 70° C.

Terminal extension activity. As used herein, terminal extension activityrefers to the ability of a reverse transcriptase (RT) to add additionalbases on to the 3′ end of a newly synthesized cDNA strand beyond the 5′end of the DNA or mRNA template. Terminal extension activity may addbases specifically (with a nucleotide bias) or randomly.

Terminal extension activity is also known as terminal deoxynucleotidyltransferase (TdT) activity. A reverse transcriptase having reduced TdTactivity is defined as any reverse transcriptase having lower TdTspecific activity than the TdT specific activity of the correspondingwild-type, unmutated, or unmodified enzyme, for example, less than about90% of the TdT specific activity of the corresponding wild-type,unmutated, or unmodified enzyme, less than about 85% of the TdT specificactivity of the corresponding wild-type, unmutated, or unmodifiedenzyme, less than about 80% of the TdT specific activity of thecorresponding wild-type, unmutated, or unmodified enzyme, less thanabout 75% of the TdT specific activity of the corresponding wild-type,unmutated, or unmodified enzyme, less than about 50% of the TdT specificactivity of the corresponding wild-type, unmutated, or unmodifiedenzyme, less than about 25% of the TdT specific activity of thecorresponding wild-type, unmutated, or unmodified enzyme, less thanabout 15% of the TdT specific activity of the corresponding wild-type,unmutated, or unmodified enzyme, less than 10% of the TdT specificactivity of the corresponding wild-type, unmutated, or unmodifiedenzyme, less than about 5% of the TdT specific activity of thecorresponding wild-type, unmutated, or unmodified enzyme, or less thanabout 1% of the TdT specific activity of the corresponding wild-type,unmutated, or unmodified enzyme. A reverse transcriptase of theinvention having substantially reduced TdT activity refers to a reversetranscriptase having a TdT specific activity level of 30% or less thanthe TdT specific activity of the corresponding wild-type or TdT⁺ reversetranscriptase. Eliminated TdT activity is defined as a level of activitythat is undetectable by the assay methods set out herein in Example 3.

As noted below in Example 3, reverse transcriptases are known in the artwhich extend nucleic acid molecules 2-3 nucleotides past the end oftemplates (e.g., RNA or DNA templates). Further, in any one reactionmixture in which reverse transcription occurs, mixtures of molecules maybe present which contain different numbers of nucleotides that extendbeyond the end of the template. TdT activity may be determined herein inreference to the number or percentage of molecules which contain one ormore nucleotides which extend beyond the end of the template. Forexample, if a wild-type reverse transcriptase adds 1 or more nucleotidespast the end of a template to 90% of the molecules generated duringreverse transcription and a modified reverse transcriptase adds 1 ormore nucleotides past the end of a template to 45% of the moleculesunder the same or similar conditions, then the modified reversetranscriptase would be said to exhibit a 50% decrease in TdT activity ascompared to the wild-type enzyme. Further, an F309N, T306K, H204R mutantof M-MLV SUPERSCRIPT™ II has been generated which exhibits about 0% ofthe TdT activity exhibited by SUPERSCRIPT™ II when DNA is used as atemplate and about 10-20% of the TdT activity exhibited by SUPERSCRIPT™II when RNA is used as a template.

Fidelity. Fidelity refers to the accuracy of polymerization, or theability of the reverse transcriptase to discriminate correct fromincorrect substrates, (e.g., nucleotides) when synthesizing nucleic acidmolecules which are complementary to a template. The higher the fidelityof a reverse transcriptase, the less the reverse transcriptasemisincorporates nucleotides in the growing strand during nucleic acidsynthesis; that is, an increase or enhancement in fidelity results in amore faithful reverse transcriptase having decreased error rate ordecreased misincorporation rate.

A reverse transcriptase having increased/enhanced/higher fidelity isdefined as a polymerase having any increase in fidelity, preferablyabout 1.2 to about 10,000 fold, about 1.5 to about 10,000 fold, about 2to about 5,000 fold, or about 2 to about 2000 fold (preferably greaterthan about 5 fold, more preferably greater than about 10 fold, stillmore preferably greater than about 50 fold, still more preferablygreater than about 100 fold, still more preferably greater than about500 fold and most preferably greater than about 100 fold) reduction inthe number of misincorporated nucleotides during synthesis of any givennucleic acid molecule of a given length compared to the control reversetranscriptase. Preferably, the mutant or modified reverse transcriptaseof the invention is compared to the corresponding unmodified orwild-type reverse transcriptase to determine the relative enhancement orincrease in fidelity. For example, a mutated reverse transcriptase maymisincorporate one nucleotide in the synthesis of a nucleic acidmolecule segment of 1000 bases compared to an unmutated reversetranscriptase misincorporating 10 nucleotides in the same size segment.Such a mutant reverse transcriptase would be said to have an increase offidelity of 10 fold.

Fidelity can also be measured by the decrease in the incidence of frameshifting, as described below in Example 5. A reverse transcriptasehaving increased fidelity may be defined as a polymerase or reversetranscriptase having any increase in fidelity with respect to frameshifting, as compared to a control reverse transcriptase (e.g., acorresponding wild-type and/or a corresponding un-mutated or un-modifiedreverse transcriptase), for example, a reverse transcriptase havinggreater than about 1.2 fold increased fidelity with respect to frameshifting, having greater than about 1.5 fold increased fidelity withrespect to frame shifting, having greater than about 5 fold increasedfidelity with respect to frame shifting, having greater than about 10fold increased fidelity with respect to frame shifting, having greaterthan about 20 fold increased fidelity with respect to frame shifting,having greater than about 30 fold increased fidelity with respect toframe shifting, or having greater than about 40 fold increased fidelitywith respect to frame shifting.

A reverse transcriptase having increased/enhanced/higher fidelity, withrespect to frame shifting, can also be defined as a reversetranscriptase or polymerase having any increase in fidelity, such asfrom about 1.5 to about 10,000 fold, from about 2 to about 5,000 fold,from about 2 to about 2000 fold, from about 1.5 to about 40 fold, fromabout 5 to about 40 fold, from about 10 to about 40 fold, from about 20to about 40 fold, from about 30 to about 40 fold, from about 5 to about30 fold, from about 10 to about 30 fold, from about 15 to about 30 fold,from about 20 to about 30 fold, from about 5 to about 20 fold, fromabout 10 to about 20 fold, from about 15 to about 20 fold, from about 10to about 100 fold, from about 15 to about 100 fold, from about 20 toabout 100 fold, from about 30 to about 100 fold, or from about 50 toabout 100 fold increased fidelity with respect to frame shifting.

A reverse transcriptase having reduced misincorporation is definedherein as either a mutated or modified reverse transcriptase that hasabout or less than 90%, has about or less than 85%, has about or lessthan 75%, has about or less than 70%, has about or less than 60%, orpreferably has about or less than 50%, preferably has about or less than25%, more preferably has about or less than 10%, and most preferably hasabout or less than 1% of relative misincorporation compared to thecorresponding wild-type, unmutated, or unmodified enzyme.

The fidelity or misincorporation rate of a reverse transcriptase can bedetermined by sequencing or by other methods known in the art (Eckert &Kunkel, 1990, Nucl. Acids Res./8:3739-3744). In one example, thesequence of a DNA molecule synthesized by the unmutated and mutatedreverse transcriptases can be compared to the expected (known) sequence.In this way, the number of errors (misincorporation or frame shifts) canbe determined for each enzyme and compared. In another example, theunmutated and mutated reverse transcriptases may be used to sequence aDNA molecule having a known sequence. The number of sequencing errors(misincorporation or frame shifts) can be compared to determine thefidelity or misincorporation rate of the enzymes. Other means ofdetermining the fidelity or misincorporation rate include a forwardcomplementation assay using an RNA template as described below andpreviously in Boyer J. C. et al. Methods Enzymol. 275:523 (1996), andare set out in the examples. Other methods of determining the fidelityor misincorporation rate will be recognized by one of skill in the art.

Strand jumping. Strand jumping, as used herein, refers to a type ofrandom mutation caused by an reverse transcriptase “skipping” more thanone (e.g., two, five, ten, fifty, one-hundred, etc.) nucleotides on themRNA template, resulting in a deletion of the corresponding nucleotidesin the resulting cDNA. Sequencing the synthesized nucleic acid moleculeand comparing to the expected sequence may allow determination of thelevel or amount of strand jumping for the reverse transcriptases of theinvention. This level or amount may then be compared to the level oramount of strand jumping caused by the corresponding wild type and/orun-modified or un-mutated reverse transcriptase.

Hand domain. The hand domain, as used herein, refers to those aminoacids which are in the area or areas that control the template, primer,or nucleotide interaction of the reverse transcriptase. This domain isfurther characterized by a group of three regions of secondary structurein a reverse transcriptase enzyme, the thumb, fingers and palm regions.The thumb region is defined as residing between amino acids 240-315 ofHIV reverse transcriptase, or between amino acids 280-355 of M-MLVreverse transcriptase. The fingers region is defined as residing betweenamino acids 1-85 and 120-154 of HIV reverse transcriptase, or between1-124 and 161-193 of M-MLV reverse transcriptase. The palm region isdefined as residing between amino acids 86-199 and 155-239 of HIVreverse transcriptase, or between amino acids 126-160 and 193-279 ofM-MLV reverse transcriptase. These areas are generally defined, and theamino acids defining the N-termini and C-termini are approximate.Corresponding regions may also be defined for other reversetranscriptases. Preferred reverse transcriptases of the invention haveone or more modifications or mutations within the hand domain. Moreparticularly, reverse transcriptases of the invention comprise one ormore mutations or modifications within one or more regions, includingthe thumb, finger, and palm regions.

Full length. As used herein, full length when used to describe a productmolecule, e.g., a cDNA molecule, indicates that the product molecule isthe same length or substantially the same length as the templatemolecule, e.g., an mRNA molecule, from which it is produced by theactivity of polypeptides of the invention. A cDNA molecule may besubstantially the same length as the template from which it is copiedwhen it is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater of the length of the portion of the template located 3′ to the3′ most nucleotide of the primer used to reverse transcribe thetemplate. Thus, if a primer anneals in the center of a template, a fulllength product would be one that contains a cDNA copy of half of thetemplate. Template molecules may be from about 100 bases to about 50 kbin length, from about 200 bases to about 50 kb in length, from about 300bases to about 50 kb in length, from about 400 bases to about 50 kb inlength, from about 500 bases to about 50 kb in length, from about 600bases to about 50 kb in length, from about 700 bases to about 50 kb inlength, from about 800 bases to about 50 kb in length, from about 900bases to about 50 kb in length, and from about 1 kb to about 50 kb inlength. In some embodiments, template molecules may be from about 500bases to about 10 kb in length, from about 600 bases to about 10 kb inlength, from about 700 bases to about 10 kb in length, from about 800bases to about 10 kb in length, from about 900 bases to about 10 kb inlength, from about 1000 bases to about 10 kb in length, from about 1100bases to about 10 kb in length, and/or from about 1200 bases to about 10kb in length. In some embodiments, template molecules may be from about250 bases to about 5 kb in length, from about 300 bases to about 5 kb inlength, and from about 350 bases to about 5 kb in length, from about 400bases to about 5 kb in length, from about 450 bases to about 5 kb inlength, from about 500 bases to about 5 kb in length, from about 550bases to about 5 kb in length, from about 600 bases to about 5 kb inlength, from about 650 bases to about 5 kb in length, from about 700bases to about 5 kb in length, from about 750 bases to about 5 kb inlength, from about 800 bases to about 5 kb in length, and from about 850bases to about 5 kb in length.

In some embodiments, the ability of a reverse transcriptase tosynthesize a full length product may be determined using a definedtemplate and primer, for example, a polyadenylated templatecorresponding to the chloramphenicol acetyl transferase gene and anoligo(dT) primer, under defined reaction conditions, e.g., pH, saltconcentration, divalent metal concentration, template concentration,temperature, etc. In some embodiments, a template molecule is greaterthan about 500 base pairs in length, and the amount of full lengthproduct synthesized may determined by separating full length productfrom truncated product, for example, by gel electrophoresis, andquantifying the full length product, for example, by incorporating aradiolabel in to the product and using a scintillation counter.

About. The term “about” as used herein, means the recited number plus orminus 10%. Thus, “about 100” includes 90-110.

Overview

In general, the invention provides, in part, compositions for use inreverse transcription of a nucleic acid molecule comprising a reversetranscriptase with one or more (e.g., one, two, three, four, five, ten,twelve, fifteen, twenty, thirty, etc.) mutations or modification whichrender the reverse transcriptase more thermostable. The invention alsoprovides compositions for use in reverse transcription of a nucleic acidmolecule, the compositions comprising a reverse transcriptase with oneor more mutations or modification which render the reverse transcriptasemore efficient, that is having higher fidelity, and/or has lower TdTactivity than a corresponding un-mutated or un-modified reversetranscriptase. The invention further provides compositions comprising areverse transcriptase with one or more mutations or modification whichrender the reverse transcriptase more thermostable and/or more efficientthan a corresponding un-mutated or un-modified reverse transcriptase.

The enzymes in these compositions are preferably present in workingconcentrations and are also preferably reduced, substantially reduced,or eliminated in RNase H activity. Alternatively, reverse transcriptasesused in the compositions of the invention may have RNase H activity.Preferred reverse transcriptases include retroviral reversetranscriptases such as M-MLV reverse transcriptase, HIV reversetranscriptase, RSV reverse transcriptase, AMV reverse transcriptase, RAVreverse transcriptase, and MAV reverse transcriptase or other ASLVreverse transcriptases or their corresponding RNase H− derivatives.Additional reverse transcriptases which may be used to preparecompositions of the invention include bacterial reverse transcriptases(e.g., Escherichia coli reverse transcriptase) (see, e.g., Mao et al.,Biochem. Biophys. Res. Commun. 227:489-93 (1996)) and reversetranscriptases of Saccharomyces cerevisiae (e.g., reverse transcriptasesof the Ty1 or Ty3 retrotransposons) (see, e.g., Cristofari et al., Jour.Biol. Chem. 274:36643-36648 (1999); Mules et al., Jour. Virol.72:6490-6503 (1998)).

In accordance with the invention, any number of mutations can be made tothe reverse transcriptases and, in a preferred aspect, multiplemutations can be made to result in an increased thermostability and/orto confer other desired properties on reverse transcriptases of theinvention. Such mutations include point mutations, frame shiftmutations, deletions and insertions, with one or more (e.g., one, two,three, four, five, ten, twelve, fifteen, twenty, thirty, etc.) pointmutations preferred. Mutations may be introduced into reversetranscriptases of the present invention using any methodology known tothose of skill in the art. Mutations may be introduced randomly by, forexample, conducting a PCR reaction in the presence of manganese as adivalent metal ion cofactor. Alternatively, oligonucleotide directedmutagenesis may be used to create the mutant polymerases which allowsfor all possible classes of base pair changes at any determined sitealong the encoding DNA molecule. In general, this technique involvesannealing an oligonucleotide complementary (except for one or moremismatches) to a single stranded nucleotide sequence coding for thereverse transcriptase of interest. The mismatched oligonucleotide isthen extended by DNA polymerase, generating a double-stranded DNAmolecule which contains the desired change in sequence in one strand.The changes in sequence can, for example, result in the deletion,substitution, or insertion of an amino acid. The double-strandedpolynucleotide can then be inserted into an appropriate expressionvector, and a mutant or modified polypeptide can thus be produced. Theabove-described oligonucleotide directed mutagenesis can, for example,be carried out via PCR.

The invention is also directed to methods for reverse transcription ofone or more (e.g., one, two, three, four, five, ten, twelve, fifteen,twenty, etc.) nucleic acid molecules comprising mixing one or more(e.g., one, two, three, four, five, ten, twelve, fifteen, twenty, etc.)nucleic acid templates, which are preferably RNA or messenger RNA (mRNA)and more preferably a population of mRNA molecules, with one or morereverse transcriptase of the present invention and incubating themixture under conditions sufficient to make a nucleic acid molecule ormolecules complementary to all or a portion of the one or more (e.g.,one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.)templates. To make the nucleic acid molecule or molecules complementaryto the one or more templates, a primer (e.g., an oligo(dT) primer) andone or more nucleotides are preferably used for nucleic acid synthesisin the 5′ to 3′ direction. Nucleic acid molecules suitable for reversetranscription according to this aspect of the invention include anynucleic acid molecule, particularly those derived from a prokaryotic oreukaryotic cell. Such cells may include normal cells, diseased cells,transformed cells, established cells, progenitor cells, precursor cells,fetal cells, embryonic cells, bacterial cells, yeast cells, animal cells(including human cells), avian cells, plant cells and the like, ortissue isolated from a plant or an animal (e.g., human, cow, pig, mouse,sheep, horse, monkey, canine, feline, rat, rabbit, bird, fish, insect,etc.). Nucleic acid molecules suitable for reverse transcription mayalso be isolated and/or obtained from viruses and/or virally infectedcells.

The invention further provides methods for amplifying or sequencing anucleic acid molecule comprising contacting the nucleic acid moleculewith a reverse transcriptase of the present invention. Preferred suchmethods comprise one or more polymerase chain reactions (PCRs).

Sources of Reverse Transcriptases

Enzymes for use in compositions, methods and kits of the inventioninclude any enzyme having reverse transcriptase activity. Such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, hepatitis B reversetranscriptase, cauliflower mosaic virus reverse transcriptase, bacterialreverse transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R.K., et al., Science 239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and4,965,188), Tne DNA polymerase (PCT Publication No. WO 96/10640), TmaDNA polymerase (U.S. Pat. No. 5,374,553) and mutants, fragments,variants or derivatives thereof (see, e.g., commonly owned U.S. Pat.Nos. 5,948,614 and 6,015,668, which are incorporated by reference hereinin their entireties). Preferably, reverse transcriptases for use in theinvention include retroviral reverse transcriptases such as M-MLVreverse transcriptase, AMV reverse transcriptase, RSV reversetranscriptase, RAV reverse transcriptase, MAV reverse transcriptase, andgenerally ASLV reverse transcriptases. As will be understood by one ofordinary skill in the art, modified reverse transcriptases may beobtained by recombinant or genetic engineering techniques that areroutine and well-known in the art. Mutant reverse transcriptases can,for example, be obtained by mutating the gene or genes encoding thereverse transcriptase of interest by site-directed or randommutagenesis. Such mutations may include point mutations, deletionmutations and insertional mutations. For example, one or more pointmutations (e.g., substitution of one or more amino acids with one ormore different amino acids) may be used to construct mutant reversetranscriptases of the invention.

The invention further includes reverse transcriptases which are 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical at the aminoacid level to a wild-type reverse transcriptase (e.g., M-MLV reversetranscriptase, AMV reverse transcriptase, RSV reverse transcriptase, HIVreverse transcriptase, etc.) and exhibit increased thermostabilityand/or other desired properties of the invention. Also included withinthe invention are reverse transcriptases which are 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical at the amino acid level to areverse transcriptase comprising the amino acid sequence set out belowin Table 3 (SEQ ID NO:2) and exhibit increased thermostability and/ormore efficient (e.g., having higher fidelity and/or having lower TdTactivity). The invention also includes nucleic acid molecules whichencode the above described reverse transcriptases.

The invention also includes fragments of reverse transcriptases whichcomprise at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or700 amino acid residues and retain one or more activities associatedwith reverse transcriptases. Such fragments may be obtained by deletionmutation, by recombinant techniques that are routine and well-known inthe art, or by enzymatic digestion of the reverse transcriptase(s) ofinterest using any of a number of well-known proteolytic enzymes. Theinvention further includes nucleic acid molecules which encode the abovedescribed mutant reverse transcriptases and reverse transcriptasefragments.

Reverse transcriptase fragments of the invention also comprise aminoacids 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90,91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160, 161-170,171-180, 181-190, 191-200, 201-210, 211-220, 221-230, 231-240, 241-250,251-260, 261-270, 271-280, 281-290, 291-300, 301-310, 311-320, 321-330,331-340, 341-350, 351-360, 361-370, 371-380, 381-390, 391-400, 401-410,411-420, 421-430, 431-440, 441-450, 451-460, 461-470, 471-480, 481-490,491-500, 501-510, 511-520, 521-530, 531-540, and/or 541-550 and/or aminoacids 1-355, 1-498, 1-500, and/or 1-550 of M-MLV reverse transcriptase(and more preferably the sequence shown in Table 3, which may furthercontain one or more of the modifications or mutations discussed herein),as well as corresponding fragments of other reverse transcriptases.Reverse transcriptase fragments of the invention further comprisepolypeptides which are 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to one or more of the fragments set out above. Theinvention also concerns various combinations of any number of thesefragments.

By a protein or protein fragment having an amino acid sequence at least,for example, 70% “identical” to a reference amino acid sequence it isintended that the amino acid sequence of the protein is identical to thereference sequence except that the protein sequence may include up to 30amino acid alterations per each 100 amino acids of the amino acidsequence of the reference protein. In other words, to obtain a proteinhaving an amino acid sequence at least 70% identical to a referenceamino acid sequence, up to 30% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 30% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino (N-) and/or carboxy (C-) terminal positions of the reference aminoacid sequence and/or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence and/or in one or more contiguous groups within the referencesequence. As a practical matter, whether a given amino acid sequence is,for example, at least 70% identical to the amino acid sequence of areference protein can be determined conventionally using known computerprograms such as those described above for nucleic acid sequenceidentity determinations, or using the CLUSTAL W program (Thompson, J.D., et al., Nucleic Acids Res. 22:4673-4680 (1994)).

Sequence identity may be determined by comparing a reference sequence ora subsequence of the reference sequence to a test sequence. Thereference sequence and the test sequence are optimally aligned over anarbitrary number of residues termed a comparison window. In order toobtain optimal alignment, additions or deletions, such as gaps, may beintroduced into the test sequence. The percent sequence identity isdetermined by determining the number of positions at which the sameresidue is present in both sequences and dividing the number of matchingpositions by the total length of the sequences in the comparison windowand multiplying by 100 to give the percentage. In addition to the numberof matching positions, the number and size of gaps is also considered incalculating the percentage sequence identity.

Sequence identity is typically determined using computer programs. Arepresentative program is the BLAST (Basic Local Alignment Search Tool)program publicly accessible at the National Center for BiotechnologyInformation (NCBI, http://www.ncbi.nlm.nih.gov/). This program comparessegments in a test sequence to sequences in a database to determine thestatistical significance of the matches, then identifies and reportsonly those matches that that are more significant than a thresholdlevel. A suitable version of the BLAST program is one that allows gaps,for example, version 2.X (Altschul, et al., Nucleic Acids Res.25(17):3389-402, 1997). Standard BLAST programs for searching nucleotidesequences (blastn) or protein (blastp) may be used. Translated querysearches in which the query sequence is translated, i.e., fromnucleotide sequence to protein (blastx) or from protein to nucleic acidsequence (tbblastn) may also be used as well as queries in which anucleotide query sequence is translated into protein sequences in all 6reading frames and then compared to an NCBI nucleotide database whichhas been translated in all six reading frames (tbblastx).

Additional suitable programs for identifying proteins with sequenceidentity to the proteins of the invention include, but are not limitedto, PHI-BLAST (Pattern Hit Initiated BLAST, Zhang, et al., Nucleic AcidsRes. 26(17):3986-90, 1998) and PSI-BLAST (Position-Specific IteratedBLAST, Altschul, et al., Nucleic Acids Res. 25(17):3389-402, 1997).

Programs may be used with default searching parameters. Alternatively,one or more search parameter may be adjusted. Selecting suitable searchparameter values is within the abilities of one of ordinary skill in theart.

Preferred enzymes for use in the invention include those that arereduced, substantially reduced, or lacking in RNase H activity. Suchenzymes that are reduced or substantially reduced in RNase H activitymay be obtained by mutating, for example, the RNase H domain within thereverse transcriptase of interest, for example, by introducing one ormore (e.g., one, two, three, four, five, ten, twelve, fifteen, twenty,thirty, etc.) point mutations, one or more (e.g., one, two, three, four,five, ten, twelve, fifteen, twenty, thirty, etc.) deletion mutations,and/or one or more (e.g., one, two, three, four, five, ten, twelve,fifteen, twenty, thirty, etc.) insertion mutations as described above.In some embodiments, the reverse transcriptase of the invention does notcontain a modification or mutation in the RNase H domain and preferablydoes not contain a modification which reduces RNase H activity. In oneaspect, the reverse transcriptase of the invention has 90%, 95%, or 100%of the RNase H activity compared to the corresponding wildtype reversetranscriptase.

By an enzyme “substantially reduced in RNase H activity” is meant thatthe enzyme has less than about 30%, less than about 25%, less than about20%, more preferably less than about 15%, less than about 10%, less thanabout 7.5%, or less than about 5%, and most preferably less than about5% or less than about 2%, of the RNase H activity of the correspondingwild-type or RNase H⁺ enzyme, such as wild-type Moloney Murine LeukemiaVirus (M-MLV), Avian Myeloblastosis Virus (AMV) or Rous Sarcoma Virus(RSV) reverse transcriptases. A reduction in RNase H activity means anyreduction in the activity compared, for example, to the correspondingwild type or un-mutatated or un-modified reverse transcriptase.

Reverse transcriptases having reduced, substantially reduced,undetectable or lacking RNase H activity have been previously described(see U.S. Pat. No. 5,668,005, U.S. Pat. No. 6,063,608, and PCTPublication No. WO 98/47912). The RNase H activity of any enzyme may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988), in Gerard, G. F., et al., FOCUS 14(5):91 (1992), in PCTpublication number WO 98/47912, and in U.S. Pat. No. 5,668,005, thedisclosures of all of which are fully incorporated herein by reference.Reverse transcriptases having no detectable RNase H activity or lackingRNase H activity by one or more of the described assays are particularlypreferred.

Particularly preferred enzymes for use in the invention include, but arenot limited to, M-MLV RNase H− reverse transcriptase, RSV RNase H−reverse transcriptase, AMV RNase H− reverse transcriptase, RAV RNase H−reverse transcriptase, MAV RNase H− reverse transcriptase and HIV RNaseH− reverse transcriptase. It will be understood by one of ordinaryskill, however, that any enzyme capable of producing a DNA molecule froma ribonucleic acid molecule (i.e., an enzyme having reversetranscriptase activity) that is reduced, substantially reduced, orlacking in RNase H activity may be equivalently used in thecompositions, methods and kits of the invention.

Enzymes for use in the invention also include those in which terminaldeoxynucleotidyl transferase (TdT) activity has been reduced,substantially reduced, or eliminated. Such enzymes that are reduced orsubstantially reduced in terminal deoxynucleotidyl transferase activity,or in which TdT activity has been eliminated, may be obtained bymutating, for example, amino acid residues within the reversetranscriptase of interest which are in close proximity or in contactwith the template-primer, for example, by introducing one or more (e.g.,one, two, three, four, five, ten, twelve, fifteen, twenty, thirty, etc.)point mutations, one or more deletion mutations, and/or one or moreinsertion mutations. Reverse transcriptases which exhibit decreased TdTactivity are described in U.S. application Ser. No. 09/808,124, filedMar. 15, 2001 (the entire disclosure of which is incorporated herein byreference), and include reverse transcriptases with one or morealterations at amino acid positions equivalent or corresponding to Y64,M289, F309, T197 and/or Y133 of M-MLV reverse transcriptase.

In one aspect, amino acid substitutions are made at one or more of theabove identified positions (i.e., amino acid positions equivalent orcorresponding to Y64, M289, F309, T197 or Y133 of M-MLV reversetranscriptase). Thus, the amino acids at these positions may besubstituted with any other amino acid including Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,and Val. Specific example of reverse transcriptases which exhibitreduced, substantially reduced, or eliminated TdT activity include M-MLVreverse transcriptases (e.g., SUPERSCRIPT™ II) in which (1) thephenylalanine residue at position 309 has been replaced with asparagine,(2) the threonine residue at position 197 has been replaced with eitheralanine or glutamic acid, and/or (3) the tyrosine residue at position133 has been replaced with alanine.

Enzymes for use in the invention also include those that exhibitincreased fidelity. Reverse transcriptases which exhibit increasedfidelity are described in U.S. Appl. No. 60/189,454, filed Mar. 15,2000, and U.S. application Ser. No. 09/808,124, filed Mar. 15, 2001 (theentire disclosures of each of which are incorporated herein byreference), and include reverse transcriptases with one or morealterations at positions equivalent or corresponding to those set outbelow in Table 2.

TABLE 2 RT Amino Acid M-MLV Y64 (e.g., Y64W, Y64R), R116 (e.g., R116M),K152 (e.g., K152R), Q190 (e.g., Q190F), T197 (e.g., T197A, T197E), V223(e.g., V223H, V223I, V223F), D124, H126, Y133 (e.g., Y133A, Y133H), F309(e.g., F309N, F309R) AMV W25, R76, K110, Q149, T156, M182 RSV W25, R76,K110, Q149, T156, M182 HIV W24, R78, G112, Q151, A158, M184

In some embodiments of the invention, amino acid substitutions are madeat one or more of the above identified positions. Thus, the amino acidsat these positions may be substituted with any other amino acidincluding Ala, Arg, Asn, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Specific example ofreverse transcriptases which exhibit increased fidelity include M-MLVreverse transcriptase in which (1) the valine residue at position 223has been replaced with histidine, phenylalanine or isoleucine, (2) thearginine residue at position 116 has been replaced with methionine, (3)the lysine residue at position 152 has been replaced with arginine, (4)the glutamic acid residue at position 190 has been replaced withphenylalanine, (5) the threonine residue at position 197 has beenreplaced with alanine or glutamic acid, (6) the phenylalanine residue atposition 309 has been replaced with asparagine or arginine, (7) thetyrosine residue at position 133 has been replaced with histidine oralanine, and/or (8) the tyrosine residue at position 64 has beenreplaced with tryptophan or arginine.

Thus, in specific embodiments, the invention includes reversetranscriptases which exhibit increased thermostability and, optionally,also exhibit one or more of the following characteristics: (1) reducedor substantially reduced RNase H activity, (2) reduced or substantiallyreduced TdT activity, and/or (3) increased fidelity.

The invention also relates to reverse transcriptase mutants, where themutations or substitutions have been made in a recognized region of thereverse transcriptase enzyme. Such regions include, but are not limitedto, the fingers, palm, thumb, α-helix H, β-sheet 10, and/or β-sheet 11regions. Thus, the invention includes reverse transcriptases whichexhibit increased thermostability (as well as other properties), asdescribed elsewhere herein, and have one or more (e.g., one, two, three,four, five, ten, fifteen, etc.) mutations or modification in the handdomain and, more specifically, in one or more regions including thefingers, palm and/or thumb regions.

Polypeptides having reverse transcriptase activity for use in theinvention may be isolated from their natural viral or bacterial sourcesaccording to standard procedures for isolating and purifying naturalproteins that are well-known to one of ordinary skill in the art (see,e.g., Houts, G. E., et al., J. Virol. 29:517 (1979); U.S. Pat. No.5,668,005; and PCT publication number WO 98/47912). In addition,polypeptides having reverse transcriptase activity may be prepared byrecombinant DNA techniques that are familiar to one of ordinary skill inthe art (see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265(1988); Soltis, D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA85:3372-3376 (1988)); U.S. Pat. No. 5,668,005; and PCT publication no.WO 98/47912.

In one aspect of the invention, mutant or modified reversetranscriptases are made by recombinant techniques. A number of clonedreverse transcriptase genes are available or may be obtained usingstandard recombinant techniques (see U.S. Pat. No. 5,668,005 and PCTPublication No. WO 98/47912).

To clone a gene or other nucleic acid molecule encoding a reversetranscriptase which will be modified in accordance with the invention,isolated, DNA which contains the reverse transcriptase gene or openreading frame may be used to construct a recombinant DNA library. Anyvector, well known in the art, can be used to clone the reversetranscriptase of interest. However, the vector used must be compatiblewith the host in which the recombinant vector will be transformed.

Prokaryotic vectors for constructing the plasmid library includeplasmids such as those capable of replication in E. coli such as, forexample, pBR322, ColE1, pSC101, pUC-vectors (pUC18, pUC19, etc.: In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); and Sambrook et al., In:Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Bacillus plasmidsinclude pC194, pUB110, pE194, pC221, pC217, etc. Such plasmids aredisclosed by Glyczan, T. In: The Molecular Biology Bacilli, AcademicPress, York (1982), 307-329. Suitable Streptomyces plasmids includepIJ101 (Kendall et al., J. Bacteriol. 169:4177-4183 (1987)). Pseudomonasplasmids are reviewed by John et al., (Rad. Insec. Dis. 8:693-704(1986)), and Igaki, (Jpn. J. Bacteriol. 33:729-742 (1978)). Broad-hostrange plasmids or cosmids, such as pCP13 (Darzins and Chakrabarty, J.Bacteriol. 159:9-18 (1984)) can also be used for the present invention.Preferred vectors for cloning the genes and nucleic acid molecules ofthe present invention are prokaryotic vectors. Preferably, pBAD, pCP13and pUC vectors are used to clone the genes of the present invention.Other suitable vectors are known to those skilled in the art and arecommercially available, for example, from Invitrogen Corporation,Carlsbad, Calif.

Suitable host for cloning the reverse transcriptase genes and nucleicacid molecules of interest are prokaryotic hosts. One example of aprokaryotic host is E. coli. However, the desired reverse transcriptasegenes and nucleic acid molecules of the present invention may be clonedin other prokaryotic hosts including, but not limited to, hosts in thegenera Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella,Serratia, and Proteus. Bacterial hosts of particular interest include E.coli DH10B, which may be obtained from Invitrogen Corporation (Carlsbad,Calif.).

Eukaryotic hosts for cloning and expression of the reverse transcriptaseof interest include yeast, fungal, and mammalian cells. Expression ofthe desired reverse transcriptase in such eukaryotic cells may requirethe use of eukaryotic regulatory regions which include eukaryoticpromoters. Cloning and expressing the reverse transcriptase gene ornucleic acid molecule in eukaryotic cells may be accomplished by wellknown techniques using well known eukaryotic vector systems.

Once a DNA library has been constructed in a particular vector, anappropriate host is transformed by well known techniques. Transformedcells are plated at a density to produce approximately 200-300transformed colonies per petri dish. For selection of reversetranscriptase, colonies are then screened for the expression of areverse transcriptase or a thermostable reverse transcriptase asdescribed in the Examples below. Briefly, overnight cultures ofindividual transformant colonies are assayed directly for reversetranscriptase or thermostable reverse transcriptase activity and/orother desirable activities using a labeled deoxynucleotide and analyzedfor the presence of labeled product. If thermostable reversetranscriptase activity and/or other desirable activity is detected, themutant is sequenced to determine which amino acids maintained reversetranscriptase activity. The gene or nucleic acid molecule encoding areverse transcriptase of the present invention can be cloned usingtechniques known to those in the art.

Modifications or Mutations of Polymerases

In accordance with the invention, one or more mutations may be made inany reverse transcriptase in order to increase the thermostability ofthe enzyme, or confer other properties described herein upon the enzyme,in accordance with the invention. Such mutations include pointmutations, frame shift mutations, deletions and insertions. Preferably,one or more point mutations, resulting in one or more amino acidsubstitutions, are used to produce reverse transcriptases havingenhanced or increased thermostability. In a preferred aspect of theinvention, one or more mutations at positions equivalent orcorresponding to position H204 (e.g., H204R) and/or T306 (e.g., T306K orT306R) of M-MLV reverse transcriptase may be made to produced thedesired result in other reverse transcriptases of interest.

In specific embodiments, one or more mutations at positions equivalentor corresponding to position L52, Y64, R116, Y133, K152 Q190, T197,H204, V223, M289, T306 and/or F309 of M-MLV reverse transcriptase may bemade to produced a desired result (e.g., increased thermostability,increased fidelity, decreased TdT activity, etc.). Thus, in specificembodiments, using amino acid positions of M-MLV reverse transcriptaseas a frame of reference, proteins of the invention include reversetranscriptases (e.g., M-MLV reverse transcriptase, AMV reversetranscriptase, HIV reverse transcriptase, RSV reverse transcriptase,etc.) having one or more of the following alterations: L52P, Y64S, Y64W,Y64R, R116M, Y133A, Y133H, K152R, K152M, Q190F, T197R, T197E, T197A,T197K, H204R, V223H, V223F, V223I, M289L, T306K, T306R, F309R, and/orF309N, as well as compositions containing these proteins, nucleic acidmolecules which encode these proteins, and host cells which containthese nucleic acid molecules.

Mutations in reverse transcriptases which alter thermostabilityproperties of these proteins may be present in conjunction withalterations which either have little or no effect on activities normallyassociated with reverse transcriptases (e.g., RNase H activity, reversetranscriptase or polymerase activity, terminal deoxynucleotidyltransferase (TdTase) activity, etc.) or substantially alter one or moreactivities normally associated with reverse transcriptases. One exampleof a reverse transcriptase which has such a combination of mutations isa M-MLV reverse transcriptase which has the following alterations:K152M, V223H.

One mutation which has been shown to enhanced the fidelity ofSUPERSCRIPT™ II (Invitrogen Corporation (Carlsbad, Calif.) Catalog No.18064-022) is V223H (see U.S. Appl. No. 60/189,454, filed Mar. 15, 2000,U.S. application Ser. No. 09/808,124, filed Mar. 15, 2001, and PCTpublication number WO 01/68895, the entire disclosures of each of whichare incorporated herein by reference). However, the V223H alterationdecreases the thermostability of this enzyme. One mutant was identified,K152M, which suppress the destabilizing effect of enzymes having theV223H mutation. Thus, the invention includes M-MLV reverse transcriptasewhich contain alterations at positions K152 and V223 and exhibit bothincreased fidelity and increased thermostability. Specific examples ofsuch reverse transcriptases are those in which K152 is replaced withmethionine and V223 is replaced with histidine. Other reversetranscriptases (e.g., AMV reverse transcriptase, HIV reversetranscriptase, RSV reverse transcriptase, etc.) with correspondingalterations are also included within the scope of the invention.

SUPERSCRIPT™ II is an RNase H-reverse transcriptase from M-MLV which hasthe following substitutions: D524G, E562Q, and D583N (see U.S. Pat. Nos.5,017,492, 5,244,797, 5,405,776, 5,668,005, and 6,063,608, the entiredisclosures of which are incorporated herein by reference). Theinvention includes reverse transcriptases that contain alterations, suchas those reference positions (i.e., 524, 562, and/or 583) or equivalentpositions.

One or more amino acid substitutions are made at one or more selectedpositions for any reverse transcriptase of interest. Thus, the aminoacids at the selected positions may be substituted with any other aminoacid including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. In some preferredembodiments, the selected amino acid will be a non-charged surfaceresidue and will be replaced by a charged residue. In some preferredembodiments, the non-charged surface residue may be replaced by apositively charged amino acid (e.g., lysine or arginine). In one aspect,a charged residue will be replaced with an un-charged residue. In oneaspect, a non-charged residue will be replaced with a negatively chargedresidue. In another aspect, a negatively charged residue will bereplaced with a positively charged residue and/or a positively chargedresidue will be replaced with a negatively charged residue.

The corresponding positions of M-MLV reverse transcriptase identifiedabove may be readily identified for other reverse transcriptases by onewith skill in the art. Thus, given the defined region and the assaysdescribed in the present application, one with skill in the art can makeone or a number of modifications which would result in increasedthermostability and/or other desired features of any reversetranscriptase of interest. Residues to be modified in accordance withthe present invention may include those listed in Table 1 above.

The nucleotide sequences for M-MLV reverse transcriptase (Shinnick etal., 1981, Nature 293:543-548; Georgiadis et al., 1995, Structure3:879-892), AMV reverse transcriptase (Joliot et al., 1993, Virology195:812-819), RSV reverse transcriptase (Schwartz et al., 1983, Cell32:853-859), and HIV reverse transcriptase (Wong-Staal et al., 1985,Nature 313:277-284) are known and are incorporated herein by referencein their entirety.

Preferably, oligonucleotide directed mutagenesis is used to create themutant reverse transcriptases which allows for all possible classes ofbase pair changes at any determined site along the encoding DNAmolecule.

Enhancing Expression of Reverse Transcriptases

To optimize expression of reverse transcriptases of the presentinvention, inducible or constitutive promoters are well known and may beused to express high levels of a reverse transcriptase structural genein a recombinant host. Similarly, high copy number vectors, well knownin the art, may be used to achieve high levels of expression. Vectorshaving an inducible high copy number may also be useful to enhanceexpression of the reverse transcriptases of the invention in arecombinant host.

To express the desired structural gene in a prokaryotic cell (such as E.coli, B. subtilis, Pseudomonas, etc.), it is preferable to operably linkthe desired structural gene to a functional prokaryotic promoter.However, the natural promoter of the reverse transcriptase gene mayfunction in prokaryotic hosts allowing expression of the reversetranscriptase gene. Thus, the natural promoter or other promoters may beused to express the reverse transcriptase gene. Such other promotersthat may be used to enhance expression include constitutive orregulatable (i.e., inducible or derepressible) promoters. Examples ofconstitutive promoters include the int promoter of bacteriophage λ, andthe bla promoter of the β-lactamase gene of pBR322. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage λ (P_(R) and P_(L)), trp, recA, lacZ, lad,tet, gal, trc, ara BAD (Guzman, et al., 1995, J. Bacteriol.177(14):4121-4130) and tac promoters of E. coli. The B. subtilispromoters include α-amylase (Ulmanen et al., J. Bacteriol 162:176-182(1985)) and Bacillus bacteriophage promoters (Gryczan, T., In: TheMolecular Biology Of Bacilli, Academic Press, New York (1982)).Streptomyces promoters are described by Ward et al., Mol. Gen. Genet.203:468478 (1986)). Prokaryotic promoters are also reviewed by Glick, J.Ind. Microbiol. 1:277-282 (1987); Cenatiempto, Y., Biochimie 68:505-516(1986); and Gottesman, Ann. Rev. Genet. 18:415-442 (1984). Expression ina prokaryotic cell also requires the presence of a ribosomal bindingsite upstream of the gene-encoding sequence. Such ribosomal bindingsites are disclosed, for example, by Gold et al., Ann. Rev. Microbiol.35:365404 (1981).

To enhance the expression of polymerases of the invention in aeukaryotic cell, well known eukaryotic promoters and hosts may be used.Enhanced expression of the polymerases may be accomplished in aprokaryotic host. One example of a prokaryotic host suitable for usewith the present invention is Escherichia coli.

Isolation and Purification of Reverse Transcriptases

The enzyme(s) of the present invention is preferably produced by growthin culture of the recombinant host containing and expressing the desiredreverse transcriptase gene. However, the reverse transcriptase of thepresent invention may be isolated from any strain, organism, or tissuewhich produces the reverse transcriptase of the present invention.Fragments of the reverse transcriptase are also included in the presentinvention. Such fragments include proteolytic fragments and fragmentshaving reverse transcriptase activity. Such fragments may also beproduced by cloning and expressing portions of the reverse transcriptasegene of interest, creating frame shift mutations and/or by adding one ormore stop codons in the gene of interest for expression of a truncatedprotein or polypeptide. Preferably, polypeptides of the invention may bepurified and/or isolated from a cell or organism expressing them, whichmay be a wild type cell or organism or a recombinant cell or organism.In some embodiments, such polypeptides may be substantially isolatedfrom the cell or organism in which they are expressed.

Any nutrient that can be assimilated by a host containing the clonedreverse transcriptase gene may be added to the culture medium. Optimalculture conditions should be selected case by case according to thestrain used and the composition of the culture medium. Antibiotics mayalso be added to the growth media to insure maintenance of vector DNAcontaining the desired gene to be expressed. Media formulations havebeen described in DSM or ATCC Catalogs and Sambrook et al., In:Molecular Cloning, a Laboratory Manual (2nd ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

Recombinant host cells producing the reverse transcriptases of thisinvention can be separated from liquid culture, for example, bycentrifugation. In general, the collected microbial cells are dispersedin a suitable buffer, and then broken open by ultrasonic treatment or byother well known procedures to allow extraction of the enzymes by thebuffer solution. After removal of cell debris by ultracentrifugation orcentrifugation, the reverse transcriptases can be purified by standardprotein purification techniques such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis or the like.Assays to detect the presence of the reverse transcriptase duringpurification are well known in the art and can be used duringconventional biochemical purification methods to determine the presenceof these enzymes.

In some embodiments, reverse transcriptases of the present invention maybe modified to contain an affinity tag in order to facilitate thepurification of the reverse transcriptase. Suitable affinity tags arewell known to those skilled in the art and include, but are not limitedto, repeated sequences of amino acids such as six histidines, epitopessuch as the hemagglutinin epitope and the myc epitope, and other aminoacid sequences that permit the simplified purification of the reversetranscriptase.

The invention further relates to fusion proteins comprising (1) aprotein, or fragment thereof, having one or more activity associatedwith a reverse transcriptase and (2) a tag (e.g., an affinity tag). Inparticular embodiments, the invention includes a reverse transcriptase(e.g., a thermostable reverse transcriptase) described herein having oneor more (e.g., one, two, three, four, five, six, seven, eight, etc.)tags. These tags may be located, for example, (1) at the N-terminus, (2)at the C-terminus, or (3) at both the N-terminus and C-terminus of theprotein, or a fragment thereof having one or more activities associatedwith a reverse transcriptase. A tag may also be located internally(e.g., between regions of amino acid sequence derived from a reversetranscriptase and/or attached to an amino acid side chain). For example,Ferguson et al., Protein Sci. 7:1636-1638 (1998), describe a siderophorereceptor, FhuA, from Escherichia coli into which an affinity tag (i.e.,a hexahistidine sequence) was inserted. This tag was shown to functionin purification protocols employing metal chelate affinitychromatography. Additional fusion proteins with internal tags aredescribed in U.S. Pat. No. 6,143,524, the entire disclosure of which isincorporated herein by reference.

Tags used to prepare compositions of the invention may vary in lengthbut will typically be from about 5 to about 500, from about 5 to about100, from about 10 to about 100, from about 15 to about 100, from about20 to about 100, from about 25 to about 100, from about 30 to about 100from about 35 to about 100, from about 40 to about 100, from about 45 toabout 100, from about 50 to about 100, from about 55 to about 100, fromabout 60 to about 100, from about 65 to about 100, from about 70 toabout 100, from about 75 to about 100, from about 80 to about 100, fromabout 85 to about 100, from about 90 to about 100, from about 95 toabout 100, from about 5 to about 80, from about 10 to about 80, fromabout 20 to about 80, from about 30 to about 80, from about 40 to about80, from about 50 to about 80, from about 60 to about 80, from about 70to about 80, from about 5 to about 60, from about 10 to about 60, fromabout 20 to about 60, from about 30 to about 60, from about 40 to about60, from about 50 to about 60, from about 5 to about 40, from about 10to about 40, from about 20 to about 40, from about 30 to about 40, fromabout 5 to about 30, from about 10 to about 30, from about 20 to about30, from about 5 to about 25, from about 10 to about 25, or from about15 to about 25 amino acid residues in length.

Tags used to prepare compositions of the invention include those whichcontribute to the thermostability of the fusion protein. Thus, thesetags may be at least partly responsible, for example, for a particularprotein (e.g., a protein having one or more activities of a reversetranscriptase activity) having increased thermostability. Examples oftags that enhance the thermostability of a reverse transcriptase (i.e.,M-MLV reverse transcriptase) include, but are not limited to, tagshaving the following amino acid sequences:MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH, which corresponds to amino acids 1-32of the sequence set forth in SEQ ID NO:2 and Table 3, andMASGTGGQQMGRDLYDDDDKH, (SEQ ID NO:3). Fragments of these tags may alsobe used in accordance with the invention (preferably those having 3 ormore, 5 or more, 10 or more, or 15 or more amino acids) Thus, theinvention includes, in part, reverse transcriptases, or fragmentsthereof that comprise tags and demonstrate enhanced thermostability.Using well known methods, one of skill in the art can attach one or moreof above-mentioned tags to one or more RT enzymes, or fragments thereofhaving reverse transcriptase activity, to produce a thermostable RTenzyme or fragment thereof. Suitable RT enzymes include, but are notlimited to, retroviral reverse transcriptase, retrotransposon reversetranscriptase, hepatitis B reverse transcriptase, cauliflower mosaicvirus reverse transcriptase, bacterial reverse transcriptase, Tth DNApolymerase, Taq DNA polymerase (Saiki, R. K., et al., Science239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNApolymerase (PCT Publication No. WO 96/10640), Tma DNA polymerase (U.S.Pat. No. 5,374,553) and mutants, fragments, variants or derivativesthereof (see, e.g., commonly owned U.S. Pat. Nos. 5,948,614 and6,015,668, which are incorporated by reference herein in theirentireties). Reverse transcriptases for use in the invention alsoinclude retroviral reverse transcriptases such as M-MLV reversetranscriptase, AMV reverse transcriptase, RSV reverse transcriptase, RAVreverse transcriptase, MAV reverse transcriptase, and generally ASLVreverse transcriptases.

Tags used in the practice of the invention may serve any number ofpurposes and a number of tags may be added to impart one or moredifferent functions to the reverse transcriptase of the invention. Forexample, tags may (1) contribute to protein-protein interactions bothinternally within a protein and with other protein molecules, (2) makethe protein amenable to particular purification methods, (3) enable oneto identify whether the protein is present in a composition; or (4) givethe protein other functional characteristics.

Examples of tags which may be used in the practice of the inventioninclude metal binding domains (e.g., a poly-histidine segments such as athree, four, five, six, or seven histidine region), immunoglobulinbinding domains (e.g., (1) Protein A; (2) Protein G; (3) T cell, B cell,and/or Fc receptors; and/or (4) complement protein antibody-bindingdomain); sugar binding domains (e.g., a maltose binding domain,chitin-binding domain); and detectable domains (e.g., at least a portionof beta-galactosidase). Fusion proteins may contain one or more tagssuch as those described above. Typically, fusion proteins that containmore than one tag will contain these tags at one terminus or bothtermini (i.e., the N-terminus and the C-terminus) of the reversetranscriptase, although one or more tags may be located internallyinstead of or in addition to those present at termini. Further, morethan one tag may be present at one terminus, internally and/or at bothtermini of the reverse transcriptase. For example, three consecutivetags could be linked end-to-end at the N-terminus of the reversetranscriptase. The invention further include compositions and reactionmixture which contain the above fusion proteins, as well as methods forpreparing these fusion proteins, nucleic acid molecules (e.g., vectors)which encode these fusion proteins and recombinant host cells whichcontain these nucleic acid molecules. The invention also includesmethods for using these fusion proteins as described elsewhere herein(e.g., methods for reverse transcribing nucleic acid molecules).

Tags which enable one to identify whether the fusion protein is presentin a composition include, for example, tags which can be used toidentify the protein in an electrophoretic gel. A number of such tagsare known in the art and include epitopes and antibody binding domainswhich can be used for Western blots.

The amino acid composition of the tags for use in the present inventionmay vary. In some embodiments, a tag may contain from about 1% to about5% amino acids that have a positive charge at physiological pH, e.g.,lysine, arginine, and histidine, or from about 5% to about 10% aminoacids that have a positive charge at physiological pH, or from about 10%to about 20% amino acids that have a positive charge at physiologicalpH, or from about 10% to about 30% amino acids that have a positivecharge at physiological pH, or from about 10% to about 50% amino acidsthat have a positive charge at physiological pH, or from about 10% toabout 75% amino acids that have a positive charge at physiological pH.In some embodiments, a tag may contain from about 1% to about 5% aminoacids that have a negative charge at physiological pH, e.g., asparticacid and glutamic acid, or from about 5% to about 10% amino acids thathave a negative charge at physiological pH, or from about 10% to about20% amino acids that have a negative charge at physiological pH, or fromabout 10% to about 30% amino acids that have a negative charge atphysiological pH, or from about 10% to about 50% amino acids that have anegative charge at physiological pH, or from about 10% to about 75%amino acids that have a negative charge at physiological pH. In someembodiments, a tag may comprise a sequence of amino acids that containstwo or more contiguous charged amino acids that may be the same ordifferent and may be of the same or different charge. For example, a tagmay contain a series (e.g., two, three, four, five, six, ten etc.) ofpositively charged amino acids that may be the same or different. A tagmay contain a series (e.g., two, three, four, five, six, ten etc.) ofnegatively charged amino acids that may be the same or different. Insome embodiments, a tag may contain a series (e.g., two, three, four,five, six, ten etc.) of alternating positively charged and negativelycharged amino acids that may be the same or different (e.g., positive,negative, positive, negative, etc.). Any of the above-described seriesof amino acids (e.g., positively charged, negatively charged oralternating charge) may comprise one or more neutral polar or non-polaramino acids (e.g., two, three, four, five, six, ten etc.) spaced betweenthe charged amino acids. Such neutral amino acids may be evenlydistributed through out the series of charged amino acids (e.g.,charged, neutral, charged, neutral) or may be unevenly distributedthroughout the series (e.g., charged, a plurality of neutral, charged,neutral, a plurality of charged, etc.). In some embodiments, tags to beattached to the polypeptides of the invention may have an overall chargeat physiological pH (e.g., positive charge or negative charge). The sizeof the overall charge may vary, for example, the tag may contain a netplus one, two, three, four, five, etc. or may possess a net negativeone, two, three, four, five, etc. The present invention also relates toreverse transcriptases (e.g., thermostable reverse transcriptases)comprising one or more of the above-described tag sequences, vectorsencoding such reverse transcriptases, host cells reaction mixture,compositions and reaction mixtures comprising such reversetranscriptases, as well as kits comprising containers containing suchreverse transcriptases.

In some embodiments, it may be desirable to remove all or a portion of atag sequence from a fusion protein comprising a tag sequence and asequence having reverse transcriptase (RT) activity. In embodiments ofthis type, one or more amino acids forming a cleavage site, e.g., for aprotease enzyme, may be incorporated into the primary sequence of thefusion protein. The cleavage site may be located such that cleavage atthe site may remove all or a portion of the tag sequence from the fusionprotein. In some embodiments, the cleavage site may be located betweenthe tag sequence and the sequence having RT activity such that all ofthe tag sequence is removed by cleavage with a protease enzyme thatrecognizes the cleavage site. Examples of suitable cleavage sitesinclude, but are not limited to, the Factor Xa cleavage site having thesequence Ile-Glu-Gly-Arg (SEQ ID NO:4), which is recognized and cleavedby blood coagulation factor Xa, and the thrombin cleavage site havingthe sequence Leu-Val-Pro-Arg (SEQ ID NO:5), which is recognized andcleaved by thrombin. Other suitable cleavage sites are known to thoseskilled in the art and may be used in conjunction with the presentinvention.

The reverse transcriptases of the invention preferably have specificactivities (e.g., RNA-directed DNA polymerase activity and/or RNase Hactivity) greater than about 5 units/mg, more preferably greater thanabout 50 units/mg, still more preferably greater than about 100units/mg, 250 units/mg, 500 units/mg, 1000 units/mg, 5000 units/mg or10,000 units/mg, and most preferably greater than about 15,000 units/mg,greater than about 16,000 units/mg, greater than about 17,000 units/mg,greater than about 18,000 units/mg, greater than about 19,000 units/mgand greater than about 20,000 units/mg. In some embodiments, the reversetranscriptases of the present invention may have specific activitiesgreater than about 50,000 units/mg, greater than about 100,000 units/mg,greater than about 150,000 units/mg, greater than about 200,000units/mg, greater than about 250,000 units/mg and greater than about300,000 units/mg. Preferred ranges of specific activities for thereverse transcriptases of the invention include a specific activity fromabout 5 units/mg to about 750,000 units/mg, a specific activity fromabout 5 units/mg to about 500,000 units/mg, from about 5 units/mg toabout 300,000 units/mg, a specific activity of from about 50 units/mg toabout 750,000 units/mg, a specific activity from about 100 units/mg toabout 750,000 units/mg, a specific activity from about 250 units/mg toabout 750,000 units/mg, a specific activity from about 500 units/mg toabout 750,000 units/mg, a specific activity from about 1000 units/mg toabout 750,000 units/mg, a specific activity from about 5000 units/mg toabout 750,000 units/mg, a specific activity from about 10,000 units/mgto about 750,000 units/mg, a specific activity from about 25,000units/mg to about 750,000 units/mg, a specific activity from about 100units/mg to about 500 units/mg, a specific activity from about 100units/mg to about 400 units/mg, and a specific activity from about 200units/mg to about 500 units/mg. Other preferred ranges of specificactivities include a specific activity of from about 200,000 units/mg toabout 350,000 units/mg, a specific activity from about 225,000 units/mgto about 300,000 units/mg, a specific activity from about 250,000units/mg to about 300,000 units/mg, a specific activity of from about200,000 units/mg to about 750,000 units/mg, a specific activity of fromabout 200,000 units/mg to about 500,000 units/mg, a specific activity offrom about 200,000 units/mg to about 400,000 units/mg, a specificactivity of from about 250,000 units/mg to about 750,000 units/mg, aspecific activity of from about 250,000 units/mg to about 500,000units/mg, and a specific activity of from about 250,000 units/mg toabout 400,000 units/mg. Preferably, the lower end of the specificactivity range may vary from 50, 100, 200, 300, 400, 500, 700, 900,1,000, 5,000, 10,000, 20,000, 30,000, 35,000, 40,000, 45,000, 50,000,55,000, 60,000, 65,000, 70,000, 75,000, and 80,000 units/mg, while theupper end of the range may vary from 750,000, 650,000, 600,000, 550,000,500,000, 450,000, 400,000, 350,000, 300,000, 250,000, 200,000, 150,000,140,000, 130,000, 120,000, 110,000, 100,000, and 90,000 units/mg.Specific activity may be determined using enzyme assays and proteinassays well known in the art. DNA polymerase assays and RNase H assaysare described, for example, in U.S. Pat. No. 5,244,797 and WO 98/47912.In some embodiments of the present invention, the specific activity ofthe thermostable reverse transcriptase prepared in accordance with thepresent invention may be higher than the specific activity of anon-thermostable reverse transcriptase. In some embodiments, thespecific activity of the thermostable reverse transcriptase may be 5%,10,%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or morehigher than the specific activity of a corresponding non-thermostablereverse transcriptase. In some preferred embodiments, the specificactivity of the thermostable reverse transcriptase according to thepresent invention may be 30% or more higher than the specific activityof a corresponding non-thermostable reverse transcriptase. In accordancewith the invention, specific activity is a measurement of the enzymaticactivity (in units) of the protein or enzyme relative to the totalamount of protein or enzyme used in a reaction. The measurement of aspecific activity may be determined by standard techniques well-known toone of ordinary skill in the art.

The reverse transcriptases of the invention may be used to make nucleicacid molecules from one or more templates. Such methods comprise mixingone or more nucleic acid templates (e.g., mRNA, and more preferably apopulation of mRNA molecules) with one or more of the reversetranscriptases of the invention and incubating the mixture underconditions sufficient to make one or more nucleic acid moleculescomplementary to all or a portion of the one or more nucleic acidtemplates.

The invention also relates to methods for the amplification of one ormore nucleic acid molecules comprising mixing one or more nucleic acidtemplates with one of the reverse transcriptases of the invention, andincubating the mixture under conditions sufficient to amplify one ormore nucleic acid molecules complementary to all or a portion of the oneor more nucleic acid templates. Such amplification methods may furthercomprise the use of one or more DNA polymerases and may be employed asin standard RT-PCR reactions.

The invention also concerns methods for the sequencing of one or morenucleic acid molecules comprising (a) mixing one or more nucleic acidmolecules to be sequenced with one or more primer nucleic acidmolecules, one or more reverse transcriptases of the invention, one ormore nucleotides and one or more terminating agents; (b) incubating themixture under conditions sufficient to synthesize a population ofnucleic acid molecules complementary to all or a portion of the one ormore nucleic acid molecules to be sequenced; and (c) separating thepopulation of nucleic acid molecules to determine the nucleotidesequence of all or a portion of the one or more nucleic acid moleculesto be sequenced.

The invention also concerns nucleic acid molecules produced by suchmethods (which may be full-length cDNA molecules), vectors (particularlyexpression vectors) comprising these nucleic acid molecules and hostcells comprising these vectors and nucleic acid molecules.

Sources of DNA Polymerase

A variety of DNA polymerases are useful in accordance with the presentinvention. Such polymerases include, but are not limited to, Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neapolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Thermococcus kodakaraensis KOD1 DNA Polymerase, Pyrococcusfuriosis (Pfu) DNA polymerase, Pyrococcus species GB-D (DEEPVENT™) DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, Mycobacterium spp. DNA polymerase (Mtb, Mlep), andmutants, variants and derivatives thereof.

DNA polymerases used in accordance with the invention may be any enzymethat can synthesize a DNA molecule from a nucleic acid template,typically in the 5′ to 3′ direction. Such polymerases may be mesophilicor thermophilic, but are preferably thermophilic. Mesophilic polymerasesinclude T5 DNA polymerase, T7 DNA polymerase, Klenow fragment DNApolymerase, DNA polymerase III, and the like. Preferred DNA polymerasesare thermostable DNA polymerases such as Taq, Tne, Tma, Pfu, VENT™,DEEPVENT™, Tth and mutants, variants and derivatives thereof (U.S. Pat.No. 5,436,149; U.S. Pat. No. 5,512,462; PCT Publication No. WO 92/06188;PCT Publication No. WO 92/06200; PCT Publication No. WO 96/10640;Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth.Appl. 2:275-287 (1993); Flaman, J.-M., et al., Nucl. Acids Res.22(15):3259-3260 (1994)). For amplification of long nucleic acidmolecules (e.g., nucleic acid molecules longer than about 3-5 Kb inlength), at least two DNA polymerases (one substantially lacking 3′exonuclease activity and the other having 3′ exonuclease activity) aretypically used. See U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462;Barnes, W. M., Gene 112:29-35 (1992); PCT Publication No. WO 98/06736;and commonly owned, co-pending U.S. patent application Ser. No.08/801,720, filed Feb. 14, 1997, the disclosures of all of which areincorporated herein in their entireties. Examples of DNA polymerasessubstantially lacking in 3′ exonuclease activity include, but are notlimited to, Taq, Tne(exo⁻), Tma, Pfu(exo⁻), Pwo and Tth DNA polymerases,and mutants, variants and derivatives thereof. Nonlimiting examples ofDNA polymerases having 3′ exonuclease activity include Pfu, DEEPVENT™and Tli/VENT™ and mutants, variants and derivatives thereof.

Formulation of Compositions and Reaction Mixtures

To form the compositions of the present invention, one or more reversetranscriptases are preferably admixed in a buffered salt solution. Oneor more DNA polymerases and/or one or more nucleotides, and/or one ormore primers may optionally be added to make the compositions of theinvention. More preferably, the enzymes are provided at workingconcentrations in stable buffered salt solutions. The terms “stable” and“stability” as used herein generally mean the retention by acomposition, such as an enzyme composition, of at least 70%, preferablyat least 80%, and most preferably at least 90%, of the originalenzymatic activity (in units) after the enzyme or composition containingthe enzyme has been stored for about one week at a temperature of about4° C., about two to six months at a temperature of about −20° C., andabout six months or longer at a temperature of about −80° C. As usedherein, the term “working concentration” means the concentration of anenzyme that is at or near the optimal concentration used in a solutionto perform a particular function (such as reverse transcription ofnucleic acids).

The water used in forming the compositions of the present invention ispreferably distilled, deionized and sterile filtered (through a 0.1-0.2micrometer filter), and is free of contamination by DNase and RNaseenzymes. Such water is available commercially, for example from SigmaChemical Company (Saint Louis, Mo.), or may be made as needed accordingto methods well known to those skilled in the art.

In addition to the enzyme components, the present compositionspreferably comprise one or more buffers and cofactors necessary forsynthesis of a nucleic acid molecule such as a cDNA molecule.Particularly preferred buffers for use in forming the presentcompositions are the acetate, sulfate, hydrochloride, phosphate or freeacid forms of Tris-(hydroxymethyl)aminomethane (TRIS®), althoughalternative buffers of the same approximate ionic strength and pKa asTRIS® may be used with equivalent results. In addition to the buffersalts, cofactor salts such as those of potassium (preferably potassiumchloride or potassium acetate) and magnesium (preferably magnesiumchloride or magnesium acetate) are included in the compositions.Addition of one or more carbohydrates and/or sugars to the compositionsand/or synthesis reaction mixtures may also be advantageous, to supportenhanced stability of the compositions and/or reaction mixtures uponstorage. Preferred such carbohydrates or sugars for inclusion in thecompositions and/or synthesis reaction mixtures of the inventioninclude, but are not limited to, sucrose, trehalose, glycerol, and thelike. Furthermore, such carbohydrates and/or sugars may be added to thestorage buffers for the enzymes used in the production of the enzymecompositions and kits of the invention. Such carbohydrates and/or sugarsare commercially available from a number of sources, including Sigma(St. Louis, Mo.).

It is often preferable to first dissolve the buffer salts, cofactorsalts and carbohydrates or sugars at working concentrations in water andto adjust the pH of the solution prior to addition of the enzymes. Inthis way, the pH-sensitive enzymes will be less subject to acid- oralkaline-mediated inactivation during formulation of the presentcompositions.

To formulate the buffered salts solution, a buffer salt which ispreferably a salt of Tris(hydroxymethyl)aminomethane (TRIS®), and mostpreferably the hydrochloride salt thereof, is combined with a sufficientquantity of water to yield a solution having a TRIS® concentration of5-150 millimolar, preferably 10-60 millimolar, and most preferably about20-60 millimolar. To this solution, a salt of magnesium (preferablyeither the chloride or acetate salt thereof) or other divalent cation,may be added to provide a working concentration thereof of 1-10millimolar, preferably 1.5-8.0 millimolar, and most preferably about3-7.5 millimolar. A salt of potassium (preferably a chloride or acetatesalt of potassium), or other monovalent cation (e.g., Na), may also beadded to the solution, at a working concentration of 10-100 millimolarand most preferably about 75 millimolar. A reducing agent, such asdithiothreitol, may be added to the solution, preferably at a finalconcentration of about 1-100 mM, more preferably a concentration ofabout 5-50 mM or about 7.5-20 mM, and most preferably at a concentrationof about 10 mM. Preferred concentrations of carbohydrates and/or sugarsfor inclusion in the compositions of the invention range from about 5%(w/v) to about 30% (w/v), from about 7.5% (w/v) to about 25% (w/v), fromabout 10% (w/v) to about 25% (w/v), from about 10% (w/v) to about 20%(w/v), and preferably from about 10% (w/v) to about 15% (w/v). A smallamount of a salt of ethylenediaminetetraacetate (EDTA), such as disodiumEDTA, may also be added (preferably about 0.1 millimolar), althoughinclusion of EDTA does not appear to be essential to the function orstability of the compositions of the present invention. After additionof all buffers and salts, this buffered salt solution is mixed welluntil all salts are dissolved, and the pH is adjusted using methodsknown in the art to a pH value of from about 7.4 to about 9.2,preferably from about 8.0 to about 9.0, and most preferably to about8.4.

To these buffered salt solutions, the enzymes (reverse transcriptasesand/or DNA polymerases) are added to produce the compositions of thepresent invention. M-MLV reverse transcriptases are preferably added ata working concentration in the solution of from about 1,000 to about50,000 units per milliliter, from about 2,000 to about 30,000 units permilliliter, from about 2,500 to about 25,000 units per milliliter, fromabout 3,000 to about 22,500 units per milliliter, from about 4,000 toabout 20,000 units per milliliter, and most preferably at a workingconcentration of from about 5,000 to about 20,000 units per milliliter.In some embodiments, a reverse transcriptases of the invention (e.g., anM-MLV reverse transcriptase) may be added at a working concentrationdescribed above in a first strand reaction mixture (e.g., a reaction toreverse transcribe an mRNA molecule) and/or in a couple RT/PCR. Asuitable concentration of a reverse transcriptase of the invention forthese reactions may be from about 5,000 units/ml to about 50,000units/ml, from about 5,000 units/ml to about 40,000 units/ml, from about5,000 units/ml to about 30,000 units/ml, or from about 5,000 units/ml toabout 20,000 units/ml of reverse transcriptase. A reaction may beconducted in a volume of 20 μl to 50 μl and such a reaction may contain50 units, 100, units, 200 units, 300 units, 400 units or more of areverse transcriptase of the invention. Those skilled in the art willappreciate that adding additional reverse transcriptase may allowincreased synthesis of the first strand (e.g., the DNA strandcomplementary to the mRNA strand) at the expense of increased enzymeusage. The skilled artisan can determine without undue experimentationthe amount of a reverse transcriptase of the invention to add to areaction in order to produce a desired amount of product at anacceptable expense.

AMV reverse transcriptases, RSV reverse transcriptases and HIV reversetranscriptases, including those of the invention described above, arepreferably added at a working concentration in the solution of fromabout 100 to about 5000 units per milliliter, from about 125 to about4000 units per milliliter, from about 150 to about 3000 units permilliliter, from about 200 to about 2500 units per milliliter, fromabout 225 to about 2000 units per milliliter, and most preferably at aworking concentration of from about 250 to about 1000 units permilliliter. The enzymes in the thermophilic DNA polymerase group (Taq,Tne, Tma, Pfu, VENT, DEEPVENT, Tth and mutants, variants and derivativesthereof) are preferably added at a working concentration in the solutionof from about 100 to about 1000 units per milliliter, from about 125 toabout 750 units per milliliter, from about 150 to about 700 units permilliliter, from about 200 to about 650 units per milliliter, from about225 to about 550 units per milliliter, and most preferably at a workingconcentration of from about 250 to about 500 units per milliliter. Theenzymes may be added to the solution in any order, or may be addedsimultaneously.

The compositions of the invention may further comprise one or morenucleotides, which are preferably deoxynucleoside triphosphates (dNTPs)or dideoxynucleoside triphosphates (ddNTPs). The dNTP components of thepresent compositions serve as the “building blocks” for newlysynthesized nucleic acids, being incorporated therein by the action ofthe polymerases, and the ddNTPs may be used in sequencing methodsaccording to the invention. Examples of nucleotides suitable for use inthe present compositions include, but are not limited to, dUTP, dATP,dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, α-thio-dATP, α-thio-dTTP,α-thio-dGTP, α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, ddGTP, ddITP,7-deaza-ddGTP, α-thio-ddATP, α-thio-ddTTP, α-thio-ddGTP, α-thio-ddCTP orderivatives thereof, all of which are available commercially fromsources including Invitrogen Corporation (Carlsbad, Calif.), New EnglandBioLabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.).The nucleotides may be unlabeled, or they may be detectably labeled bycoupling them by methods known in the art with radioisotopes (e.g., ³H,¹⁴C, ³²P or ³⁵S), vitamins (e.g., biotin), fluorescent moieties (e.g.,fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescentlabels (e.g., using the PHOTO-GENE™ or ACES™ chemiluminescence systems,available commercially from Invitrogen Corporation (Carlsbad, Calif.)),dioxigenin and the like. Labeled nucleotides may also be obtainedcommercially, for example from Invitrogen Corporation (Carlsbad, Calif.)or Sigma Chemical Company (Saint Louis, Mo.). In the presentcompositions, the nucleotides are added to give a working concentrationof each nucleotide of about 10-4000 micromolar, about 50-2000micromolar, about 100-1500 micromolar, or about 200-1200 micromolar, andmost preferably a concentration of about 1000 micromolar.

To reduce component deterioration, storage of the reagent compositionsis preferably at about 4° C. for up to one day, or most preferably at−20° C. for up to one year.

In another aspect, the compositions and reverse transcriptases of theinvention may be prepared and stored in dry form in the presence of oneor more carbohydrates, sugars, or synthetic polymers. Preferredcarbohydrates, sugars or polymers for the preparation of driedcompositions or reverse transcriptases include, but are not limited to,sucrose, trehalose, and polyvinylpyrrolidone (PVP) or combinationsthereof. See, e.g., U.S. Pat. Nos. 5,098,893, 4,891,319, and 5,556,771,the disclosures of which are entirely incorporated herein by reference.Such dried compositions and enzymes may be stored at varioustemperatures for extended times without significant deterioration ofenzymes or components of the compositions of the invention. Preferably,the dried reverse transcriptases or compositions are stored at 4° C. orat −20° C.

The invention further includes reaction solutions for reversetranscribing nucleic acid molecules, as well as reverse transcriptionmethods employing such reaction solutions and product nucleic acidmolecules produced using such methods. In many instances, reactionsolutions of the invention will contain one or more of the followingcomponents: (1) one or more buffering agent (e.g., sodium phosphate,sodium acetate, 2-(N-morpholino)-ethanesulfonic acid (MES),tris-(hydroxymethyl)aminomethane (Tris),3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPS), citrate,N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), acetate,3-(N-morpholino)propanesulfonic acid (MOPS),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonio acid (TAPS), etc.),(2) one or more monovalent cationic salt (e.g., NaCl, KCl, etc.), (3)one or more divalent cationic salt (e.g., MnCl₂, MgCl₂, MgSO₄, CaCl₂,etc.), (4) one or more reducing agent (e.g., dithiothreitol,β-mercaptoethanol, etc.), (5) one or more ioninc or non-ionic detergent(e.g., TRITON X-100™, NONIDET P40™, sodium dodecyl sulphate, etc.), (6)one or more DNA polymerase inhibitor (e.g., Actinomycin D, etc.), (7)nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.), (8) RNAto be reverse transcribed and/or amplified, (9) one or more RNaseinhibitor (e.g., RNASEOUT™, Invitrogen Corporation, Carlsbad, Calif.,catalog number 10777-019 etc.), (10) a reverse transcriptase (e.g., areverse transcriptase of the invention, and/or (11) one or more diluent(e.g., water). Other components and/or constituents (e.g., primers, DNApolymerases, etc.) may also be present in reaction solutions.

The concentration of the buffering agent in the reaction solutions ofthe invention will vary with the particular buffering agent used.Typically, the working concentration (i.e., the concentration in thereaction mixture) of the buffering agent will be from about 5 mM toabout 500 mM (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM,about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM,about 85 mM, about 90 mM, about 95 mM, about 100 mM, from about 5 mM toabout 500 mM, from about 10 mM to about 500 mM, from about 20 mM toabout 500 mM, from about 25 mM to about 500 mM, from about 30 mM toabout 500 mM, from about 40 mM to about 500 mM, from about 50 mM toabout 500 mM, from about 75 mM to about 500 mM, from about 100 mM toabout 500 mM, from about 25 mM to about 50 mM, from about 25 mM to about75 mM, from about 25 mM to about 100 mM, from about 25 mM to about 200mM, from about 25 mM to about 300 mM, etc.). When Tris (e.g., Tris-HCl)is used, the Tris working concentration will typically be from about 5mM to about 100 mM, from about 5 mM to about 75 mM, from about 10 mM toabout 75 mM, from about 10 mM to about 60 mM, from about 10 mM to about50 mM, from about 25 mM to about 50 mM, etc.

The final pH of solutions of the invention will generally be set andmaintained by buffering agents present in reaction solutions of theinvention. The pH of reaction solutions of the invention, and hencereaction mixtures of the invention, will vary with the particular useand the buffering agent present but will often be from about pH 5.5 toabout pH 9.0 (e.g., about pH 6.0, about pH 6.5, about pH 7.0, about pH7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, from aboutpH 6.0 to about pH 8.5, from about pH 6.5 to about pH 8.5, from about pH7.0 to about pH 8.5, from about pH 7.5 to about pH 8.5, from about pH6.0 to about pH 8.0, from about pH 6.0 to about pH 7.7, from about pH6.0 to about pH 7.5, from about pH 6.0 to about pH 7.0, from about pH7.2 to about pH 7.7, from about pH 7.3 to about pH 7.7, from about pH7.4 to about pH 7.6, from about pH 7.0 to about pH 7.4, from about pH7.6 to about pH 8.0, from about pH 7.6 to about pH 8.5, from about pH7.7 to about pH 8.5, from about pH 7.9 to about pH 8.5, from about pH8.0 to about pH 8.5, from about pH 8.2 to about pH 8.5, from about pH8.3 to about pH 8.5, from about pH 8.4 to about pH 8.5, from about pH8.4 to about pH 9.0, from about pH 8.5 to about pH 9.0, etc.)

As indicated, one or more monovalent cationic salts (e.g., NaCl, KCl,etc.) may be included in reaction solutions of the invention. In manyinstances, salts used in reaction solutions of the invention willdissociate in solution to generate at least one species which ismonovalent (e.g., Na+, K+, etc.) When included in reaction solutions ofthe invention, salts will often be present either individually or in acombined concentration of from about 0.5 mM to about 500 mM (e.g., about1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 12 mM,about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM,about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 64 mM, about65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM,about 95 mM, about 100 mM, about 120 mM, about 140 mM, about 150 mM,about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM,about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM,from about 1 mM to about 500 mM, from about 5 mM to about 500 mM, fromabout 10 mM to about 500 mM, from about 20 mM to about 500 mM, fromabout 30 mM to about 500 mM, from about 40 mM to about 500 mM, fromabout 50 mM to about 500 mM, from about 60 mM to about 500 mM, fromabout 65 mM to about 500 mM, from about 75 mM to about 500 mM, fromabout 85 mM to about 500 mM, from about 90 mM to about 500 mM, fromabout 100 mM to about 500 mM, from about 125 mM to about 500 mM, fromabout 150 mM to about 500 mM, from about 200 mM to about 500 mM, fromabout 10 mM to about 100 mM, from about 10 mM to about 75 mM, from about10 mM to about 50 mM, from about 20 mM to about 200 mM, from about 20 mMto about 150 mM, from about 20 mM to about 125 mM, from about 20 mM toabout 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about75 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM,from about 30 mM to about 500 mM, from about 30 mM to about 100 mM, fromabout 30 mM to about 70 mM, from about 30 mM to about 50 mM, etc.).

As indicated, one or more divalent cationic salts (e.g., MnCl₂, MgCl₂,MgSO₄, CaCl₂, etc.) may be included in reaction solutions of theinvention. In many instances, salts used in reaction solutions of theinvention will dissociate in solution to generate at least one specieswhich is monovalent (e.g., Mg⁺⁺, Mn⁺⁺, Ca⁺⁺, etc.) When included inreaction solutions of the invention, salts will often be present eitherindividually or in a combined concentration of from about 0.5 mM toabout 500 mM (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM,about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM,about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM,about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about64 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM,about 90 mM, about 95 mM, about 100 mM, about 120 mM, about 140 mM,about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM,about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM,about 400 mM, from about 1 mM to about 500 mM, from about 5 mM to about500 mM, from about 10 mM to about 500 mM, from about 20 mM to about 500mM, from about 30 mM to about 500 mM, from about 40 mM to about 500 mM,from about 50 mM to about 500 mM, from about 60 mM to about 500 mM, fromabout 65 mM to about 500 mM, from about 75 mM to about 500 mM, fromabout 85 mM to about 500 mM, from about 90 mM to about 500 mM, fromabout 100 mM to about 500 mM, from about 125 mM to about 500 mM, fromabout 150 mM to about 500 mM, from about 200 mM to about 500 mM, fromabout 10 mM to about 100 mM, from about 10 mM to about 75 mM, from about10 mM to about 50 mM, from about 20 mM to about 200 mM, from about 20 mMto about 150 mM, from about 20 mM to about 125 mM, from about 20 mM toabout 100 mM, from about 20 mM to about 80 mM, from about 20 mM to about75 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM,from about 30 mM to about 500 mM, from about 30 mM to about 100 mM, fromabout 30 mM to about 70 mM, from about 30 mM to about 50 mM, etc.).

When included in reaction solutions of the invention, reducing agents(e.g., dithiothreitol, β-mercaptoethanol, etc.) will often be presenteither individually or in a combined concentration of from about 0.1 mMto about 50 mM (e.g., about 0.2 mM, about 0.3 mM, about 0.5 mM, about0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM,about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about 17mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM,about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about50 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 50mM, from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, fromabout 3 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 0.5mM to about 2.5 mM, from about 1 mM to about 20 mM, from about 1 mM toabout 10 mM, from about 1 mM to about 5 mM, from about 1 mM to about 3.4mM, from about 0.5 mM to about 3.0 mM, from about 1 mM to about 3.0 mM,from about 1.5 mM to about 3.0 mM, from about 2 mM to about 3.0 mM, fromabout 0.5 mM to about 2.5 mM, from about 1 mM to about 2.5 mM, fromabout 1.5 mM to about 2.5 mM, from about 2 mM to about 3.0 mM, fromabout 2.5 mM to about 3.0 mM, from about 0.5 mM to about 2 mM, fromabout 0.5 mM to about 1.5 mM, from about 0.5 mM to about 1.1 mM, fromabout 5.0 mM to about 10 mM, from about 5.0 mM to about 15 mM, fromabout 5.0 mM to about 20 mM, from about 10 mM to about 15 mM, from about10 mM to about 20 mM, etc.).

Reaction solutions of the invention may also contain one or more ionincor non-ionic detergent (e.g., TRITON X-100™, NONIDET P40™, sodiumdodecyl sulphate, etc.). When included in reaction solutions of theinvention, detergents will often be present either individually or in acombined concentration of from about 0.01% to about 5.0% (e.g., about0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%,about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.9%, about 1%, about2%, about 3%, about 4%, about 5%, from about 0.01% to about 5.0%, fromabout 0.01% to about 4.0%, from about 0.01% to about 3.0%, from about0.01% to about 2.0%, from about 0.01% to about 1.0%, from about 0.05% toabout 5.0%, from about 0.05% to about 3.0%, from about 0.05% to about2.0%, from about 0.05% to about 1.0%, from about 0.1% to about 5.0%,from about 0.1% to about 4.0%, from about 0.1% to about 3.0%, from about0.1% to about 2.0%, from about 0.1% to about 1.0%, from about 0.1% toabout 0.5%, etc.). For example, reaction solutions of the invention maycontain TRITON X100™ at a concentration of from about 0.01% to about2.0%, from about 0.03% to about 1.0%, from about 0.04% to about 1.0%,from about 0.05% to about 0.5%, from about 0.04% to about 0.6%, fromabout 0.04% to about 0.3%, etc.

Reaction solutions of the invention may also contain one or more DNApolymerase inhibitor (e.g., Actinomycin D, etc.). When included inreaction solutions of the invention, such inhibitors will often bepresent either individually or in a combined concentration of from about0.1 μg/ml to about 100 μg/ml (e.g., about 0.1 μg/ml, about 0.2 μg/ml,about 0.3 μg/ml, about 0.4 μg/ml, about 0.5 μg/ml, about 0.6 μg/ml,about 0.7 μg/ml, about 0.8 μg/ml, about 0.9 μg/ml, about 1.0 μg/ml,about 1.1 μg/ml, about 1.3 μg/ml, about 1.5 μg/ml, about 1.7 μg/ml,about 2.0 μg/ml, about 2.5 μg/ml, about 3.5 μg/ml, about 5.0 μg/ml,about 7.5 μg/ml, about 10 μg/ml, about 15 μg/ml, about 20 μg/ml, about25 μg/ml, about 30 μg/ml, about 35 μg/ml, about 40 μg/ml, about 50μg/ml, about 60 μg/ml, about 70 μg/ml, about 80 μg/ml, about 90 μg/ml,about 100 μg/ml, from about 0.5 μg/ml to about 30 μg/ml, from about 0.75μg/ml to about 30 μg/ml, from about 1.0 μg/ml to about 30 μg/ml, fromabout 2.0 μg/ml to about 30 μg/ml, from about 3.0 μg/ml to about 30μg/ml, from about 4.0 μg/ml to about 30 μg/ml, from about 5.0 μg/ml toabout 30 μg/ml, from about 7.5 μg/ml to about 30 μg/ml, from about 10μg/ml to about 30 μg/ml, from about 15 μg/ml to about 30 μg/ml, fromabout 0.5 μg/ml to about 20 μg/ml, from about 0.5 μg/ml to about 10μg/ml, from about 0.5 μg/ml to about 5 μg/ml, from about 0.5 μg/ml toabout 2 μg/ml, from about 0.5 μg/ml to about 1 μg/ml, from about 1 μg/mlto about 10 μg/ml, from about 1 μg/ml to about 5 μg/ml, from about 1μg/ml to about 2 μg/ml, from about 1 μg/ml to about 100 μg/ml, fromabout 10 μg/ml to about 100 μg/ml, from about 20 μg/ml to about 100μg/ml, from about 40 μg/ml to about 100 μg/ml, from about 30 μg/ml toabout 80 μg/ml, from about 30 μg/ml to about 70 μg/ml, from about 40μg/ml to about 60 μg/ml, from about 40 μg/ml to about 70 μg/ml, fromabout 40 μg/ml to about 80 μg/ml, etc.).

In many instances, nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP,dTTP, etc.) will be present in reaction mixtures of the invention.Typically, individually nucleotides will be present in concentrations offrom about 0.05 mM to about 50 mM (e.g., about 0.07 mM, about 0.1 mM,about 0.15 mM, about 0.18 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM,about 0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4mM, about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM,about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about50 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 50mM, from about 1 mM to about 50 mM, from about 2 mM to about 50 mM, fromabout 3 mM to about 50 mM, from about 0.5 mM to about 20 mM, from about0.5 mM to about 10 mM, from about 0.5 mM to about 5 mM, from about 0.5mM to about 2.5 mM, from about 1 mM to about 20 mM, from about 1 mM toabout 10 mM, from about 1 mM to about 5 mM, from about 1 mM to about 3.4mM, from about 0.5 mM to about 3.0 mM, from about 1 mM to about 3.0 mM,from about 1.5 mM to about 3.0 mM, from about 2 mM to about 3.0 mM, fromabout 0.5 mM to about 2.5 mM, from about 1 mM to about 2.5 mM, fromabout 1.5 mM to about 2.5 mM, from about 2 mM to about 3.0 mM, fromabout 2.5 mM to about 3.0 mM, from about 0.5 mM to about 2 mM, fromabout 0.5 mM to about 1.5 mM, from about 0.5 mM to about 1.1 mM, fromabout 5.0 mM to about 10 mM, from about 5.0 mM to about 15 mM, fromabout 5.0 mM to about 20 mM, from about 10 mM to about 15 mM, from about10 mM to about 20 mM, etc.). The combined nucleotide concentration, whenmore than one nucleotides is present, can be determined by adding theconcentrations of the individual nucleotides together. When more thanone nucleotide is present in reaction solutions of the invention, theindividual nucleotides may not be present in equimolar amounts. Thus, areaction solution may contain, for example, 1 mM dGTP, 1 mM dATP, 0.5 mMdCTP, and 1 mM dTTP.

RNA will typically be present in reaction solutions of the invention. Inmost instances, RNA will be added to the reaction solution shortly priorto reverse transcription. Thus, reaction solutions may be providedwithout RNA. This will typically be the case when reaction solutions areprovided in kits. RNA, when present in reaction solutions will often bepresent in a concentration of 1 picogram to 100 μg/20 μl reactionmixture (e.g., about 1 picogram/20 μl, about 10 picograms/20 μl, about50 picograms/20 μl, about 100 picograms/20 μl, about 200 picograms/20μl, about 10 picograms/20 μl, about 500 picograms/20 μl, about 800picograms/20 μl, about 1.0 nanogram/20 μl, about 5.0 nanograms/20 μl,about 10 nanograms/20 μl, about 25 nanograms/20 μl, about 50nanograms/20 μl, about 75 nanograms/20 μl, about 100 nanograms/20 μl,about 150 nanograms/20 μl, about 250 nanograms/20 μl, about 400nanograms/20 μl, about 500 nanograms/20 μl, about 750 nanograms/20 μl,about 1.0 μg/20 about 5.0 μg/20 μl, about 10 μg/20 μl, about 20 μg/20μl, about 30 μg/20 μl, about 40 μg/20 μl, about 50 μg/20 μl, about 70μg/20 μl, about 85 μg/20 μl, about 100 μg/20 μl, from about 10picograms/20 μl to about 100 μg/20 μl, from about 10 picograms/20 μl toabout 100 μg/20 μl, from about 100 picograms/20 μl to about 100 μg/20μl, from about 1.0 nanograms/20 μl to about 100 μg/20 μl, from about 100nanograms/20 μl to about 100 μg/20 μl, from about 10 picograms/20 μl toabout 10 μg/20 μl, from about 10 picograms/20 μl to about 5 μg/20 μl,from about 100 nanograms/20 μl to about 5 μg/20 μl, from about 1 μg/20μl to about 10 μg/20 μl, from about 1 μg/20 μl to about 5 μg/20 μl, fromabout 100 nanograms/20 μl to about 1 μg/20 μl, from about 500nanograms/20 μl to about 5 μg/20 μl, etc.). As one skilled in the artwould recognize, different reverse transcription reactions may beperformed in volumes other than 20 μl. In such instances, the totalamount of RNA present will vary with the volume used. Thus, the aboveamounts are provided as examples of the amount of RNA/20 μl of reactionsolution.

Reverse transcriptases (e.g., reverse transcriptases of the invention)may also be present in reaction solutions. When present, reversetranscriptases, will often be present in a concentration which resultsin about 0.01 to about 1,000 units of reverse transcriptase activity/μl(e.g., about 0.01 unit/μl, about 0.05 unit/μl, about 0.1 unit/μl, about0.2 unit/μl, about 0.3 unit/μl, about 0.4 unit/μl, about 0.5 unit/μl,about 0.7 unit/μl, about 1.0 unit/μl, about 1.5 unit/μl, about 2.0unit/μl, about 2.5 unit/μl, about 5.0 unit/μl, about 7.5 unit/μl, about10 unit/μl, about 20 unit/μl, about 25 unit/μl, about 50 unit/μl, about100 unit/μl, about 150 unit/μl, about 200 unit/μl, about 250 unit/μl,about 350 unit/μl, about 500 unit/μl, about 750 unit/μl, about 1,000unit/μl, from about 0.1 unit/μl to about 1,000 unit/μl, from about 0.2unit/μl to about 1,000 unit/μl, from about 1.0 unit/μl to about 1,000unit/μl, from about 5.0 unit/μl to about 1,000 unit/μl, from about 10unit/μl to about 1,000 unit/μl, from about 20 unit/μl to about 1,000unit/μl, from about 50 unit/μl to about 1,000 unit/μl, from about 100unit/μl to about 1,000 unit/μl, from about 200 unit/μl to about 1,000unit/μl, from about 400 unit/μl to about 1,000 unit/μl, from about 500unit/μl to about 1,000 unit/μl, from about 0.1 unit/μl to about 300unit/μl, from about 0.1 unit/μl to about 200 unit/μl, from about 0.1unit/μl to about 100 unit/μl, from about 0.1 unit/μl to about 50unit/μl, from about 0.1 unit/μl to about 10 unit/μl, from about 0.1unit/μl to about 5.0 unit/μl, from about 0.1 unit/μl to about 1.0unit/μl, from about 0.2 unit/μl to about 0.5 unit/μl, etc.

Reaction solutions of the invention may be prepared as concentratedsolutions (e.g., 5× solutions) which are diluted to a workingconcentration for final use. With respect to a 5× reaction solution, a5:1 dilution is required to bring such a 5× solution to a workingconcentration. Reaction solutions of the invention may be prepared, forexamples, as a 2×, a 3×, a 4×, a 5×, a 6×, a 7×, a 8×, a 9×, a 10×, etc.solutions. One major limitation on the fold concentration of suchsolutions is that, when compounds reach particular concentrations insolution, precipitation occurs. Thus, concentrated reaction solutionswill generally be prepared such that the concentrations of the variouscomponents are low enough so that precipitation of buffer componentswill not occur. As one skilled in the art would recognize, the upperlimit of concentration which is feasible for each solution will varywith the particular solution and the components present.

In many instances, reaction solutions of the invention will be providedin sterile form. Sterilization may be performed on the individualcomponents of reaction solutions prior to mixing or on reactionsolutions after they are prepared. Sterilization of such solutions maybe performed by any suitable means including autoclaving orultrafiltration.

Labeling Nucleic Acids

In general, the invention provides, in part, compositions for use inreverse transcription of a nucleic acid molecule to produce labelednucleic acid molecules. Such compositions may comprise one or morereverse transcriptases (e.g., single subunit and/or multi-subunit RTs).The enzymes in these compositions are preferably present in workingconcentrations and have RNase H activity or are reduced or substantiallyreduced or lacking in RNase H activity, although mixtures of enzymes,some having RNase H activity and some reduced or substantially reducedor lacking in RNase H activity, may be used in the compositions of theinvention. Preferred reverse transcriptases include M-MLV reversetranscriptase, RSV reverse transcriptase, AMV reverse transcriptase, RAVreverse transcriptase, MAV reverse transcriptase and HIV reversetranscriptase or other ASLV reverse transcriptases.

The invention is also directed to methods for reverse transcription ofone or more nucleic acid molecules comprising mixing one or more nucleicacid templates, which is preferably RNA or messenger RNA (mRNA) and morepreferably a population of mRNA molecules, with one or more polypeptideshaving reverse transcriptase activity and incubating the mixture underconditions sufficient to make one or more labeled nucleic acid moleculescomplementary to all or a portion of the one or more templates. To makethe nucleic acid molecule or molecules complementary to the one or moretemplates, at least one primer (e.g., an oligo(dT) primer) and one ormore nucleotides (a portion of which are preferably labeled, mostpreferably fluorescently labeled) are used for nucleic acid synthesis.Nucleic acid templates suitable for reverse transcription according tothis aspect of the invention include any nucleic acid molecule,particularly those derived from a prokaryotic or eukaryotic cell. Suchcells may include normal cells, diseased cells, transformed cells,established cells, progenitor cells, precursor cells, fetal cells,embryonic cells, bacterial cells, yeast cells, animal cells (includinghuman cells), avian cells, plant cells and the like, or tissue isolatedfrom a plant or an animal (e.g., human, cow, pig, mouse, sheep, horse,monkey, canine, feline, rat, rabbit, bird, fish, insect, etc.). Suchnucleic acid molecules may also be isolated from viruses. In someembodiments, methods of the invention result in the direct labeling of anucleic acid molecule by incorporation of a labeled nucleotide (e.g., anucleotide containing a fluorescent label). In other methods, nucleicacid molecules are indirectly labeled by first, incorporating anucleotide analog containing a reactive functionality to produce anucleic acid containing one or more reactive functionalities. Thenucleic acid containing reactive functionalities may subsequently bereacted with a molecule containing a label that reacts with thefunctionality to attach the label to the nucleic acid molecule. Thereaction may be result in covalent attachment of all or a portion of thelabel-containing molecule to the nucleic acid molecule (e.g., chemicalcoupling). In some embodiments, amine-modified NTPs (e.g., aminoallyl-dUTP/UTP) are incorporated during reverse transcription. Aminoallyl-NTPs are incorporated with similar efficiency as unmodified NTPsduring enzymatic reactions such as reverse transcription. The aminefunctionality is then coupled with a dye using standard techniques. Kitsfor indirect labeling of cDNA are commercially available from, forexample, Ambion, Inc., Austin, Tex. In some embodiments, thelabel-containing molecule may be non-covalently bound to the reactivefunctionality. For example, the reactive functionality may be a biotinmoiety and the label-containing molecule may be a labeled (e.g.,fluorescently labeled) avidin or streptavidin molecule.

The invention also provides labeled nucleic acid molecules producedaccording to the above-described methods. Such labeled nucleic acidmolecules may be single or double stranded and are useful as detectionprobes. Depending on the labeled nucleotide(s) used during synthesis,the labeled molecules may contain one or a number of labels. Wheremultiple labels are used, the molecules may comprise a number of thesame or different labels. Thus, one type or multiple different labelednucleotides may be used during synthesis of nucleic acid molecules toprovide for the labeled nucleic acid molecules of the invention. Suchlabeled nucleic acid molecules will thus comprise one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, etc.) labelednucleotides (which may be the same or different).

Labeled nucleic acid molecules produced by methods of the invention mayeither (1) comprise a particular numbers of labeled nucleotides or (2) aparticular percentage of the nucleotides present in the nucleic acidmolecule may be labeled. In either instance, the concentration oflabeled nucleotides present in the reaction mixture may be adjusted,with respect to un-labeled nucleotides, such that product nucleic acidmolecules are produced which contain (1) a particular number of labelednucleotides or (2) a particular percentage of label nucleotides ascompared to un-labeled nucleotides. In particular instances, from about0.1% to about 20%, from about 0.1% to about 15%, from about 0.1% toabout 10%, from about 0.1% to about 5.0%, from about 0.1% to about 2.5%,from about 0.1% to about 1.5%, from about 0.1% to about 1.0%, from about0.1% to about 0.5%, from about 2.0% to about 20%, from about 4.0% toabout 20%, from about 0.5% to about 10%, from about 0.5% to about 5%,from about 0.5% to about 2.0%, or from about 0.5% to about 1.0% of thetotal nucleotides present in product nucleic acid molecules are labeled.As one skilled in the art would recognize, the actual number of labelednucleotides, and thus the percentage of nucleotides which are labeled,present in individual molecules of a population will typically differ.In other words, different members of populations of nucleic acidmolecules will typically contain different numbers of labelednucleotides with the overall average of labeled nucleotides present ineach product molecule varying with a number of factors (e.g., the ratioof labeled to un-labeled nucleotides present in the reaction mixture).In most instances, at least 85%, at least 90%, at least 95%, or at least99% of the individual product nucleic acid molecules in the populationwill contain labeled nucleotides which fall within a range set outabove.

In accordance with the invention, the amount of labeled product ispreferably measured based on percent incorporation of the label ofinterest into synthesized product as may be determined by one skilled inthe art, although other means of measuring the amount or efficiency oflabeling of product will be recognized by one of ordinary skill in theart. The invention provides for enhanced or increased percentincorporation of labeled nucleotide during synthesis of a nucleic acidmolecule from a template, preferably during synthesis of one or morecDNA molecules from RNA. According to the invention, such enhancement orincrease in percent incorporation is preferably about equal to orgreater than a 2-fold, a 5-fold, a 10-fold, a 15-fold, a 20-fold, a25-fold, a 30-fold, a 40-fold or a 50-fold increase or enhancement inpercent incorporation compared to a standard reverse transcriptase.

The invention also provides kits for use in accordance with theinvention. Such kits comprise a carrier means, such as a box or carton,having in close confinement therein one or more container means, such asvials, tubes, bottles and the like, wherein the kit comprises, in thesame or different containers, one or more reverse transcriptases. Thekits of the invention may also comprise, in the same or differentcontainers, one or more DNA polymerases, one or more primers, one ormore suitable buffers and/or one or more nucleotides (such asdeoxynucleoside triphosphates (dNTPs) and preferably labeled dNTPs(e.g., fluorescently labeled dNTPs)).

In some embodiments, the RTs used in the invention comprise two or moresubunits (or derivatives, variants, fragments or mutants thereof) andpreferably comprise two subunits (e.g., a dimer or heterodimer). Twosubunit reverse transcriptases typically have an α and a β subunitforming a dimer, although any form or combination of subunits (andderivatives, variants or mutants of such subunits) may be used. Suchcombinations may include αβ, ββ, αα and the like. Preferred two subunitRTs for use in the invention include RSV RT, AMV RT, AEV RT, RAV RT, HIVRT and MAV RT, or other ASLV RTs, or mutants, variants or derivativesthereof. In a particular embodiment, AMV RT and/or RSV RT is used inaccordance with the invention. Preferred single subunit RTs includeM-MLV reverse transcriptase.

Production/Sources of cDNA Molecules

In accordance with the invention, cDNA molecules (single-stranded ordouble-stranded) may be prepared from a variety of nucleic acid templatemolecules. Preferred nucleic acid molecules for use in the presentinvention include single-stranded or double-stranded DNA and RNAmolecules, as well as double-stranded DNA:RNA hybrids. More preferrednucleic acid molecules include messenger RNA (mRNA), transfer RNA (tRNA)and ribosomal RNA (rRNA) molecules, although mRNA molecules are thepreferred template according to the invention.

The nucleic acid molecules that are used to prepare cDNA moleculesaccording to the methods of the present invention may be preparedsynthetically according to standard organic chemical synthesis methodsthat will be familiar to one of ordinary skill. More preferably, thenucleic acid molecules may be obtained from natural sources, such as avariety of cells, tissues, organs or organisms. Cells that may be usedas sources of nucleic acid molecules may be prokaryotic (bacterialcells, including but not limited to those of species of the generaEscherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic(including fungi (especially yeasts), plants, protozoans and otherparasites, and animals including insects (particularly Drosophila spp.cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human cells)).

Mammalian somatic cells that may be used as sources of nucleic acidsinclude blood cells (reticulocytes and leukocytes), endothelial cells,epithelial cells, neuronal cells (from the central or peripheral nervoussystems), muscle cells (including myocytes and myoblasts from skeletal,smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells. Also suitable for use as nucleic acidsources are mammalian tissues or organs such as those derived frombrain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous,skin, genitourinary, circulatory, lymphoid, gastrointestinal andconnective tissue sources, as well as those derived from a mammalian(including human) embryo or fetus.

Any of the above prokaryotic or eukaryotic cells, tissues and organs maybe normal, diseased, transformed, established, progenitors, precursors,fetal or embryonic. Diseased cells may, for example, include thoseinvolved in infectious diseases (caused by bacteria, fungi or yeast,viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, muscular dystrophy ormultiple sclerosis) or in cancerous processes. Transformed orestablished animal cell lines may include, for example, COS cells, CHOcells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293cells, L929 cells, F9 cells, and the like. Other cells, cell lines,tissues, organs and organisms suitable as sources of nucleic acids foruse in the present invention will be apparent to one of ordinary skillin the art.

Once the starting cells, tissues, organs or other samples are obtained,nucleic acid molecules (such as mRNA) may be isolated therefrom bymethods that are well-known in the art (See, e.g., Maniatis, T., et al.,Cell 15:687-701 (1978); Okayama, H., and Berg, P., Mol. Cell. Biol.2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene 25:263-269(1983)). The nucleic acid molecules thus isolated may then be used toprepare cDNA molecules and cDNA libraries in accordance with the presentinvention.

In the practice of the invention, cDNA molecules or cDNA libraries areproduced by mixing one or more nucleic acid molecules obtained asdescribed above, which is preferably one or more mRNA molecules such asa population of mRNA molecules, with a polypeptide having reversetranscriptase activity of the present invention, or with one or more ofthe compositions of the invention, under conditions favoring the reversetranscription of the nucleic acid molecule by the action of the enzymesor the compositions to form one or more cDNA molecules (single-strandedor double-stranded). Thus, the method of the invention comprises (a)mixing one or more nucleic acid templates (preferably one or more RNA ormRNA templates, such as a population of mRNA molecules) with one or morereverse transcriptases of the invention and (b) incubating the mixtureunder conditions sufficient to make one or more nucleic acid moleculescomplementary to all or a portion of the one or more templates. Suchmethods may include the use of one or more DNA polymerases, one or morenucleotides, one or more primers, one or more buffers, and the like. Theinvention may be used in conjunction with methods of cDNA synthesis suchas those described in the Examples below, or others that are well-knownin the art (see, e.g., Gubler, U., and Hoffman, B. J., Gene 25:263-269(1983); Krug, M. S., and Berger, S. L., Meth. Enzymol. 152:316-325(1987); Sambrook, J., et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press,pp. 8.60-8.63 (1989); PCT Publication No. WO 99/15702; PCT PublicationNo. WO 98/47912; and PCT Publication No. WO 98/51699), to produce cDNAmolecules or libraries.

Other methods of cDNA synthesis which may advantageously use the presentinvention will be readily apparent to one of ordinary skill in the art.

Having obtained cDNA molecules or libraries according to the presentmethods, these cDNAs may be isolated for further analysis ormanipulation. Detailed methodologies for purification of cDNAs aretaught in the GENETRAPPER™ manual (Invitrogen Corporation (Carlsbad,Calif.)), which is incorporated herein by reference in its entirety,although alternative standard techniques of cDNA isolation that areknown in the art (see, e.g., Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 8.60-8.63 (1989)) may also be used.

In other aspects of the invention, the invention may be used in methodsfor amplifying and sequencing nucleic acid molecules. Nucleic acidamplification methods according to this aspect of the invention may beone-step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR)reactions. According to the invention, one-step RT-PCR type reactionsmay be accomplished in one tube thereby lowering the possibility ofcontamination. Such one-step reactions comprise (a) mixing a nucleicacid template (e.g., mRNA) with one or more reverse transcriptases ofthe present invention and with one or more DNA polymerases and (b)incubating the mixture under conditions sufficient to amplify a nucleicacid molecule complementary to all or a portion of the template. Suchamplification may be accomplished by the reverse transcriptase activityalone or in combination with the DNA polymerase activity. Two-stepRT-PCR reactions may be accomplished in two separate steps. Such amethod comprises (a) mixing a nucleic acid template (e.g., mRNA) with areverse transcriptase of the present invention, (b) incubating themixture under conditions sufficient to make a nucleic acid molecule(e.g., a DNA molecule) complementary to all or a portion of thetemplate, (c) mixing the nucleic acid molecule with one or more DNApolymerases, and (d) incubating the mixture of step (c) under conditionssufficient to amplify the nucleic acid molecule. For amplification oflong nucleic acid molecules (i.e., greater than about 3-5 Kb in length),a combination of DNA polymerases may be used, such as one DNA polymerasehaving 3′ exonuclease activity and another DNA polymerase beingsubstantially reduced in 3′ exonuclease activity.

Nucleic acid sequencing methods according to this aspect of theinvention may comprise both cycle sequencing (sequencing in combinationwith amplification) and standard sequencing reactions. The sequencingmethod of the invention thus comprises (a) mixing a nucleic acidmolecule to be sequenced with one or more primers, one or more reversetranscriptases of the invention, one or more nucleotides and one or moreterminating agents, (b) incubating the mixture under conditionssufficient to synthesize a population of nucleic acid moleculescomplementary to all or a portion of the molecule to be sequenced, and(c) separating the population to determine the nucleotide sequence ofall or a portion of the molecule to be sequenced. According to theinvention, one or more DNA polymerases (preferably thermostable DNApolymerases) may be used in combination with or separate from thereverse transcriptases of the invention.

Amplification methods which may be used in accordance with the presentinvention include PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202), StrandDisplacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0 684 315),and Nucleic Acid Sequence-Based Amplification (NASBA; U.S. Pat. No.5,409,818; EP 0 329 822), as well as more complex PCR-based nucleic acidfingerprinting techniques such as Random Amplified Polymorphic DNA(RAPD) analysis (Williams, J. G. K., et al., Nucl. Acids Res.18(22):6531-6535, 1990), Arbitrarily Primed PCR (AP-PCR; Welsh, J., andMcClelland, M., Nucl. Acids Res. 18(24):7213-7218, 1990), DNAAmplification Fingerprinting (DAF; Caetano-Anollés et al.,Bio/Technology 9:553-557, 1991), microsatellite PCR or DirectedAmplification of Minisatellite-region DNA (DAMD; Heath, D. D., et al.,Nucl. Acids Res. 21(24): 5782-5785, 1993), and Amplification FragmentLength Polymorphism (AFLP) analysis (EP 0 534 858; Vos, P., et al.,Nucl. Acids Res. 23(21):4407-4414, 1995; Lin, J. J., and Kuo, J., FOCUS17(2):66-70, 1995). Nucleic acid sequencing techniques which may employthe present compositions include dideoxy sequencing methods such asthose disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523. In aparticularly preferred aspects, the invention may be used in methods ofamplifying or sequencing a nucleic acid molecule comprising one or morepolymerase chain reactions (PCRs), such as any of the PCR-based methodsdescribed above.

Kits

In another embodiment, the present invention may be assembled into kits,which may be used in reverse transcription or amplification of a nucleicacid molecule, or into kits for use in sequencing of a nucleic acidmolecule. Kits according to this aspect of the invention comprise acarrier means, such as a box, carton, tube or the like, having in closeconfinement therein one or more container means, such as vials, tubes,ampoules, bottles and the like, wherein a first container means containsone or more polypeptides of the present invention having reversetranscriptase activity. When more than one polypeptide having reversetranscriptase activity is used, they may be in a single container asmixtures of two or more polypeptides, or in separate containers. Thekits of the invention may also comprise (in the same or separatecontainers) one or more DNA polymerases, a suitable buffer, one or morenucleotides and/or one or more primers. The kits of the invention mayalso comprise one or more hosts or cells including those that arecompetent to take up nucleic acids (e.g., DNA molecules includingvectors). Preferred hosts may include chemically competent orelectrocompetent bacteria such as E. coli (including DH5, DH5α, DH10B,HB101, Top 10, and other K-12 strains as well as E. coli B and E. coli Wstrains).

In a specific aspect of the invention, the kits of the invention (e.g.,reverse transcription and amplification kits) may comprise one or morecomponents (in mixtures or separately) including one or morepolypeptides having reverse transcriptase activity of the invention, oneor more nucleotides (one or more of which may be labeled, e.g.,fluorescently labeled) used for synthesis of a nucleic acid molecule,and/or one or more primers (e.g., oligo(dT) for reverse transcription).Such kits (including the reverse transcription and amplification kits)may further comprise one or more DNA polymerases. Sequencing kits of theinvention may comprise one or more polypeptides having reversetranscriptase activity of the invention, and optionally one or more DNApolymerases, one or more terminating agents (e.g., dideoxynucleosidetriphosphate molecules) used for sequencing of a nucleic acid molecule,one or more nucleotides and/or one or more primers. Preferredpolypeptides having reverse transcriptase activity, DNA polymerases,nucleotides, primers and other components suitable for use in thereverse transcription, amplification and sequencing kits of theinvention include those described above. The kits encompassed by thisaspect of the present invention may further comprise additional reagentsand compounds necessary for carrying out standard nucleic acid reversetranscription, amplification or sequencing protocols. Such polypeptideshaving reverse transcriptase activity of the invention, DNA polymerases,nucleotides, primers, and additional reagents, components or compoundsmay be contained in one or more containers, and may be contained in suchcontainers in a mixture of two or more of the above-noted components ormay be contained in the kits of the invention in separate containers.Such kits may also comprise instructions (e.g., for performing themethods of the invention such as for labeling nucleic acid molecules inaccordance with the invention).

Use of Nucleic Acid Molecules

The nucleic acid molecules or cDNA libraries prepared by the methods ofthe present invention may be further characterized, for example bycloning and sequencing (i.e., determining the nucleotide sequence of thenucleic acid molecule), by the sequencing methods of the invention or byothers that are standard in the art (see, e.g. U.S. Pat. Nos. 4,962,022and 5,498,523, which are directed to methods of DNA sequencing).Alternatively, these nucleic acid molecules may be used for themanufacture of various materials in industrial processes, such ashybridization probes by methods that are well-known in the art.Production of hybridization probes from cDNAs will, for example, providethe ability for those in the medical field to examine a patient's cellsor tissues for the presence of a particular genetic marker such as amarker of cancer, of an infectious or genetic disease, or a marker ofembryonic development. Furthermore, such hybridization probes can beused to isolate DNA fragments from genomic DNA or cDNA librariesprepared from a different cell, tissue or organism for furthercharacterization.

The nucleic acid molecules of the present invention may also be used toprepare compositions for use in recombinant DNA methodologies.Accordingly, the present invention relates to recombinant vectors whichcomprise the cDNA or amplified nucleic acid molecules of the presentinvention, to host cells which are genetically engineered with therecombinant vectors, to methods for the production of a recombinantpolypeptide using these vectors and host cells, and to recombinantpolypeptides produced using these methods.

Recombinant vectors may be produced according to this aspect of theinvention by inserting, using methods that are well-known in the art,one or more of the cDNA molecules or amplified nucleic acid moleculesprepared according to the present methods into a vector. The vector usedin this aspect of the invention may be, for example, a phage or aplasmid, and is preferably a plasmid. Preferred are vectors comprisingcis-acting control regions to the nucleic acid encoding the polypeptideof interest. Appropriate trans-acting factors may be supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression (and are therefore termed “expression vectors”),which may be inducible and/or cell type-specific. Particularly preferredamong such vectors are those inducible by environmental factors that areeasy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids, and will preferablyinclude at least one selectable marker such as a tetracycline orampicillin resistance gene for culturing in a bacterial host cell. Priorto insertion into such an expression vector, the cDNA or amplifiednucleic acid molecules of the invention should be operatively linked toan appropriate promoter, such as the phage lambda P_(L) promoter, the E.coli lac, trp and tac promoters. Other suitable promoters will be knownto the skilled artisan.

Among vectors preferred for use in the present invention include pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; pcDNA3 available from Invitrogen; pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available fromPharmacia; and pSPORT1, pSPORT2 and pSV.SPORT1, available fromInvitrogen Corporation (Carlsbad, Calif.). Other suitable vectors willbe readily apparent to the skilled artisan.

The invention also provides methods of producing a recombinant host cellcomprising the cDNA molecules, amplified nucleic acid molecules orrecombinant vectors of the invention, as well as host cells produced bysuch methods. Representative host cells (prokaryotic or eukaryotic) thatmay be produced according to the invention include, but are not limitedto, bacterial cells, yeast cells, plant cells and animal cells.Preferred bacterial host cells include Escherichia coli cells (mostparticularly E. coli strains DH10B and Stb12, which are availablecommercially (Invitrogen Corporation (Carlsbad, Calif.)), Bacillussubtilis cells, Bacillus megaterium cells, Streptomyces spp. cells,Erwinia spp. cells, Klebsiella spp. cells and Salmonella typhimuriumcells. Preferred animal host cells include insect cells (mostparticularly Spodoptera frugiperda Sf9 and Sf21 cells and TrichoplusaHigH-Five cells) and mammalian cells (most particularly CHO, COS, VERO,BHK and human cells). Such host cells may be prepared by well-knowntransformation, electroporation or transfection techniques that will befamiliar to one of ordinary skill in the art.

In addition, the invention provides methods for producing a recombinantpolypeptide, and polypeptides produced by these methods. According tothis aspect of the invention, a recombinant polypeptide may be producedby culturing any of the above recombinant host cells under conditionsfavoring production of a polypeptide therefrom, and isolation of thepolypeptide. Methods for culturing recombinant host cells, and forproduction and isolation of polypeptides therefrom, are well-known toone of ordinary skill in the art.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1 Preparation of Mutant Reverse Transcriptases

Plasmid pBAD was obtained from Invitrogen Corporation, Carlsbad, Calif.and the coding sequence of M-MLV reverse transcriptase was inserted toproduce plasmid pBAD-6-His-M-MLV H− (F1). Plasmid pBAD-6-His-M-MLV H−(F1) was used as both a cloning vector and as a target for PCRmutagenesis (FIG. 1). pBAD-6-His-M-MLV H− (F1) replicates in E. coli andconfers ampicillin resistance to transformed cells. The M-MLV reversetranscriptase gene is expressed from the ara BAD promoter which isinduced by the presence of arabinose. The promoter is repressed by theproduct of the araC gene, which is present on the plasmid. The hostused, E. coli strain DH10B, is an araD mutant and cannot metabolizearabinose, making arabinose a gratuitous inducer in DH10B cellstransformed with pBAD-6-His-M-MLV H−(F1). The plasmid contains a 6histidine containing leader sequence in frame with the coding sequenceof the M-MLV reverse transcriptase gene. The gene starting at nucleotide2598 and ending at nucleotide 4628 (Shinnick, et al., (1981) Nature 293,543-548.) was cloned under control of an araD promoter into plasmidpBAD/H isA (Invitrogen). The M-MLV gene was further modified bysite-directed mutagenesis without changing amino acid coding to includeseveral unique restriction endonuclease sites that divided the gene intofive segments (FIG. 2). The amino end of the protein contained a His₆tag to simplify purification that included the following amino acids:MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH corresponding to amino acids 1-32 ofSEQ ID NO:2. The carboxy end of the protein contained the additionalamino acids NSRLIN, corresponding to amino acids 711-716 of SEQ ID NO:2,present as the result of subcloning from pRT601. In addition, the M-MLVRT gene was mutated (D524G, E562Q, D583N) to eliminate RNase H activity.The final construct was designated pBAD-HSS2 (FIG. 1), and the gene andgene product were designated His₆ H-RT. In addition to this construct,other constructs having different N-terminal sequences are contemplatedin the present invention. For example, a construct beginning atmethionine 12 of SEQ ID NO:6 and Table 3 and containing a mutationchanging methionine 15 to glycine (M15G) to produce a protein with anN-terminal sequence MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:1)-followed by theremaining sequence of M-MLV RT from Table 3 has been produced as well asa construct beginning with methionine 33 of SEQ ID NO:6 and Table 3.

With reference to the sequence of this plasmid provided in Table 3 (SEQID NOs:1 and 2), nucleotides 1-96 encode the leader sequence andnucleotides 97-99 encode a methionine. Those skilled in the art willappreciate that the wild-type M-MLV reverse transcriptase is derived byproteolysis from a precursor polyprotein and thus the wild-type M-MLVreverse transcriptase does not start with a methionine. Therefore, aminoacid number 1 of the M-MLV reverse transcriptase is the threonine (aminoacid 34 in SEQ ID NO:2 and Table 3) following the methionine encoded bynucleotides 97-99 (amino acid 33 in SEQ ID NO:2 and Table 3).

The sequence of the M-MLV reverse transcriptase gene in pBAD-6-His-M-MLVH− (F1) which was used in these experiments was derived from thesequence of plasmid pRT601. pRT601 is described in Kotewicz, et al.,(1988) Nuc. Acids Res. 16, 265-277, Gerard, et al., (1986) DNA 5,271-279, U.S. Pat. Nos. 5,668,005 and 5,017,492, which are incorporatedherein by reference in their entireties.

TABLE 3 (SEQ ID NOs: 1 and 2). 1atggggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa  m  g  g   s  h  h   h  h  h  h   g  m  a   s  m  t   g  g  q  q 61atgggtcggg atctgtacga cgatgacgat aagcatatga ccctaaatat agaagatgag  m  g  r   d  l  y   d  d  d  d   k  h  m   t  l  n   i  e  d  e 121tatcggctac atgagacctc aaaagagcca gatgtttctc tagggtccac atggctgtct  y  r  l   h  e  t   s  k  e  p   d  v  s   l  g  s   t  w  l  s 181gattttcctc aggcctgggc ggaaaccggg ggcatgggac tggcagttcg ccaagctcct  d  f  p   q  a  w   a  e  t  g   g  m  g   l  a  v   r  q  a  p 241ctgatcatac ttctgaaagc aacctctacc cccgtgtcca taaaacaata ccccatgtca  l  i  i   l  l  k   a  t  s  t   p  v  s   i  k  q   y  p  m  s 301caagaagcca gactggggat caagccccac atacagagac tgttggacca gggaatactg  q  e  a   r  l  g   i  k  p  h   i  q  r   l  l  d   q  g  i  l 361gtaccctgcc agtccccctg gaacacgccc ctgctacccg tcaagaaacc cgggactaat  v  p  c   q  s  p   w  n  t  p   l  l  p   v  k  k   p  g  t  n 421gattacaggc ctgtccaaga tctgagagag gtcaacaaac gcgtagaaga catccacccc  d  y  r   p  v  q   d  l  r  e   v  n  k   r  v  e   d  i  h  p 481accgtaccca acccctacaa cctcttgagt gggctcccac cgtcccacca gtggtacact  t  v  p   n  p  y   n  l  l  s   g  l  p   p  s  h   q  w  y  t 541gttctagact taaaagatgc ctttttctgc ctgagactcc acccgacgtc tcagcctctc  v  l  d   l  k  d   a  f  f  c   l  r  l   h  p  t   s  q  p  l 601ttcgcctttg aatggagaga cccagagatg ggaatctctg gccaactaac ctggaccaga  f  a  f   e  w  r   d  p  e  m   g  i  s   g  q  l   t  w  t  r 661ctcccacagg gattcaaaaa cagtcccacc ctgtttgatg aggcactgcg cagagaccta  l  p  q   g  f  k   n  s  p  t   l  f  d   e  a  l   r  r  d  l 721gcagacttcc ggatccagca cccagacttg atcctgctac agtacgtaga tgacttactg  a  d  f   r  i  q   h  p  d  l   i  l  l   q  y  v   d  d  l  l 781ctggccgcca cttctgagct cgactgccaa caaggtactc gggccctgtt acaaacccta  l  a  a   t  s  e   l  d  c  q   q  g  t   r  a  l   l  q  t  l 841ggagacctcg ggtatcgggc ctcggccaag aaagcccaaa tttgccagaa acaggtcaag  g  d  l   g  y  r   a  s  a  k   k  a  q   i  c  q   k  q  v  k 901tatctggggt atcttctaaa agagggtcag agatggctga ctgaggccag aaaagagact  y  l  g   y  l  l   k  e  g  q   r  w  l   t  e  a   r  k  e  t 961gtgatggggc agcctactcc gaagaccccg cggcaactaa gggagttcct agggacggca  v  m  g   q  p  t   p  k  t  p   r  q  l   r  e  f   l  g  t  a 1021ggcttctgtc gcctctggat ccctgggttt gcagaaatgg cagccccctt gtaccctctc  g  f  c   r  l  w   i  p  g  f   a  e  m   a  a  p   l  y  p  l 1081accaaaacgg ggactctgtt taattggggc ccagaccaac aaaaggccta tcaagaaatc  t  k  t   g  t  l   f  n  w  g   p  d  q   q  k  a   y  q  e  i 1141aagcaagctc ttctaactgc cccagccctg gggttgccag atttgactaa gccctttgaa  k  q  a   l  l  t   a  p  a  l   g  l  p   d  l  t   k  p  f  e 1201ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc taacgcaaaa actgggacct  l  f  v   d  e  k   q  g  y  a   k  g  v   l  t  q   k  l  g  p 1261tggcgtcggc cggtggccta cctgtccaaa aagctagacc cagtagcagc tgggtggccc  w  r  r   p  v  a   y  l  s  k   k  l  d   p  v  a   a  g  w  p 1321ccttgcctac ggatggtagc agccattgcc gtactgacaa aggatgcagg caagctaacc  p  c  l   r  m  v   a  a  i  a   v  l  t   k  d  a   g  k  l  t 1381atgggacagc cactagtcat tctggccccc catgcagtag aggcactagt caaacaaccc  m  g  q   p  l  v   i  l  a  p   h  a  v   e  a  l   v  k  q  p 1441cccgatcgat ggctttccaa cgcccggatg actcactatc aggccttgct tttggacacg  p  d  r   w  l  s   n  a  r  m   t  h  y   q  a  l   l  l  d  t 1501gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg ctacactgct cccactgcct  d  r  v   q  f  g   p  v  v  a   l  n  p   a  t  l   l  p  l  p 1561gaggaagggc tgcagcacaa ctgccttgat atcctggccg aagcccacgg aacccgaccc  e  e  g   l  q  h   n  c  l  d   i  l  a   e  a  h   g  t  r  p 1621gacctaacgg accagccgct cccagacgcc gaccacacct ggtacacggg tggatccagt  d  l  t   d  q  p   l  p  d  a   d  h  t   w  y  t   g  g  s  s 1681ctcttgcaag agggacagcg taaggcggga gctgcggtga ccaccgagac cgaggtaatc  l  l  q   e  g  q   r  k  a  g   a  a  v   t  t  e   t  e  v  i 1741tgggctaaag ccctgccagc cgggacatcc gctcagcggg ctcagctgat agcactcacc  w  a  k   a  l  p   a  g  t  s   a  q  r   a  q  l   i  a  l  t 1801caggccctaa ggatggcaga aggtaagaag ctaaatgttt atacgaattc ccgttatgct  q  a  l   r  m  a   e  g  k  k   l  n  v   y  t  n   s  r  y  a 1861tttgctactg cccatatcca tggagaaata tacagaaggc gtgggttgct cacatcagaa  f  a  t   a  h  i   h  g  e  i   y  r  r   r  g  l   l  t  s  e 1921ggcaaagaga tcaaaaataa ggacgagata ttggccctac taaaagccct ctttctgccc  g  k  e   i  k  n   k  d  e  i   l  a  l   l  k  a   l  f  l  p 1981aaaagactta gcataatcca ttgtccagga catcaaaagg gacacagcgc cgaggctaga  k  r  l   s  i  i   h  c  p  g   h  q  k   g  h  s   a  e  a  r 2041ggcaaccgga tggctgacca agcggcccga aaggcagcca tcacagagaa tccagacacc  g  n  r   m  a  d   q  a  a  r   k  a  a   i  t  e   n  p  d  t 2101tctaccctcc tcatagaaaa ttcatcaccc aattcccgct taattaatta a  s  t  l   l  i  e   n  s  s  p   n  s  r   l  i  n   -

Table 4 provides a list of the point mutations introduced in the M-MLVreverse transcriptase coding sequence of pRT601 to produce the plasmidused. The numbering of the point mutations corresponds to the nucleotidesequence presented in Table 3.

TABLE 4 Nucleotide # in Table 3 change 411 a_(→)c 459 g_(→)a 462 g_(→)c543 g_(→)t 546 t_(→)a 585 c_(→)g 588 c_(→)g 589 a_(→)t 590 g_(→)c 639a_(→)t 642 a_(→)c 710 a_(→)g 801 a_(→)c 990 t_(→)g 993 a_(→)g 1446c_(→)t 1449 c_(→)a 1670 a_(→)g 1675 a_(→)t 1676 g_(→)c 1783 g_(→)c 1785a_(→)g 1845 t_(→)g 1846 g_(→)a 1849 a_(→)t 1850 g_(→)c 1950 c_(→)a

The mutations which were introduced to make RNase H-mutants of M-MLVreverse transcriptase are D524G, D583N, and E562Q. The remainingmutations were introduced to insert or remove restriction enzyme sitesto facilitate the production of appropriately sized segments for therandom PCR mutagenesis. This RNase H-mutant is referred to herein asSUPERSCRIPT™ II or SUPERSCRIPT™ II gene.

The sequence of the M-MLV reverse transcriptase was engineered tointroduce restriction enzyme cleavage sites as shown schematically inFIG. 2 without changing the amino acids encoded by the sequence. Thesequence was divided into 5 segments and oligonucleotides were designedso that each segment could be amplified. The segments roughlycorresponded to the coding sequences of the five separate structuralsubdomains of RT (Kohlstaedt, et al., (1992) Science 256, 1783-1789,Jacobo-Molina, et al., (1993) Proc. Natl. Acad. Sci. USA 90, 6320-6324).Segments one through four corresponded to the polymerase subdomain offingers, palm, thumb, and connection, respectively, and segment fivecorresponded to the RNase H domain (FIG. 2). An upper limit cut-off of 1to 2 mutations per segment was set as the target for mutation frequencyto suppress accumulation of deleterious mutations, and to minimize theamount of screening required to find active mutants. Mutationfrequencies of >5 mutations/segment in segment one, two, or threeproduced only about 5% active mutants with all the mutants having lessthan wild-type activity. At the mutation frequency used of 1 to 2mutations per segment, approximately one-third of the mutants had littleor no activity, one-third had less than 50% of His₆ H-RT activity, andone-third had up to 100% of His₆ H-RT activity.

Segments were prepared from pBAD-6-His-M-MLV H− (F1) by restrictionenzyme digests and the segments were gel purified away from the vectorbackbone. Each segment was randomly mutagenized by PCR in the presenceof manganese. The PCR conditions were standard except that 0.25 mM MnCl₂was present, and the nucleotide triphosphate concentration was limitedto 20 μM of each dNTP (50 mM Tris.HCl pH 8.3, 50 mM KCl, 3 mM MgCl₂, 20μM dGTP, 20 μM dCTP, 20 μM dATP, 20 μM dTTP, 1 unit Taq DNA polymeraseper 100 μl reaction). The PCR product was extracted withphenol-chloroform, precipitated with ethanol and the mutated segmentswere cloned into a vector from which the given segment had been removed.

In some random mutagenesis experiments, mutagenic PCR was performed in areaction mixture (100 μl) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl,1.8 mM MgCl₂, 0.3 mM MnSO₄, 200 μM each of dCTP, dGTP, dTTP, and dATP,and 0.5 units of Taq DNA polymerase (Invitrogen Corporation, Carlsbad,Calif.). After a 1 min denaturation step at 94° C., the cycling protocolwas 15 sec at 94° C., 15 sec at 55° C., and 30 sec at 72° C. for 20cycles. Amplification was 100 fold from 50 ng of target to 5 μg ofamplified product. PCR primers included appropriate restrictionendonuclease cut sites. An amplified DNA segment was cleaved withappropriate restriction endonucleases, gel purified, and cloned into gelpurified vector DNA cut with the corresponding restriction enzymes. Thevectors containing the mutated segments were transformed intoappropriate host cells to produce a library.

Libraries were sampled by DNA sequencing of the mutagenized H-RT genesegment of a small number of clones to determine the mutation frequency.The goal was to achieve rates of PCR random mutagenesis that produced 1to 2 mutations per segment. It was found that random mutagenesis thatproduced greater than 2 mutations per segment tended to produce a largeproportion of inactive RT mutants. If the library met this criterion,further screening by heat treatment was carried out to identify mutantsthat showed greater RT activity in lysates than H− RT after a heattreatment step. Those mutants with the highest apparent thermalstability were screened again in duplicate by pretreatment at 24° C. (tonormalize for activity) and at 52 to 58° C. to confirm the presence ofenhanced thermal stability. In all, about 15,000 clones were screened byheat treatment in the 96-well format for each segment or a total ofabout 100,000 mutants. Libraries of transformants for each mutatedsegment were screened for thermostable variants.

Example 2 Screening for Thermostable Reverse Transcriptases

In this example the following solutions were used:

EG-per liter: 20 g bacto-tryptone, 10 g bacto yeast extract, 2 mlglycerol, 0.54 g NaCl, 0.194 g KCl

EG-arabinose-150 ml EG plus 1.5 ml of 10 mg/ml ampicillin and 1.5 ml of20% (w/v) arabinose (if plates are to have arabinose)

20×PEB-I Buffer-18% (w/v) glucose, 500 mM Tris-HCl (pH 8.0), 200 mM EDTA

Kinase Storage Buffer-50% (v/v) glycerol, 20 mM Tris-HCl (pH 8.0), 100mM KCl, 5 mM βME

100 mg/ml lysozyme-made in Kinase Storage Buffer and stored at −20° C.

2×PLD-5 ml of 20×PEB-I, 1 ml of 1 M DTT, 5 ml of 10% (v/v) Triton X-100,1 ml of 100 mg/ml lysozyme and 38 ml of water

2×PZD-0.5 ml of 20×PEB-I, 100 μl of 1 M DTT, 0.5 ml of 10% (v/v) TritonX-100, 10 μl of zymolase and 3.9 ml of water

10× Poly(C) Reaction Buffer-500 mM Tris-HCl (pH 8.4), 500 mM KCl, 100 mMMgCl₂

1.25× Reaction Mix-1 ml of 10× Poly(C) Reaction Buffer, 100 μl of 1 MDTT, 1 ml of poly(C)/oligo(dG) (30 mM/12 mM in nucleotide), 10 μl of 100mM dGTP, 5.87 ml of water and 20 μl of [α-³²P] dGTP at 10 μCi/μl

E. coli DH10B (Invitrogen Corporation, Carlsbad, Calif.) was used in allexperiments. Bacterial liquid cultures were grown at 37° C. in EG: 2%tryptone, 1% yeast extract, 0.5% glycerol, 10 mM NaCl, and 1 mM KCl.Solid medium was LB (1% bacto-tryptone, 0.5% yeast extract, and 86 mMNaCl)+1.5% agar. Selective media included 100 μg/ml ampicillin. Forinduction of cultures, cells were inoculated into selective EG+0.2%arabinose and grown for 18 hr.

Mutant populations were plated on selective agar. Individualtransformant colonies were inoculated into single wells of a 96 wellculture plate. Each well contained 120 μl of EG-Ap medium (EG mediumwith 100 μg/ml ampicillin). Although colonies from the selective agarmay be grown in media containing 0.2% arabinose, it is preferable tofirst inoculate a 96 well plate with selective medium without theinducer, to grow that master plate overnight, and then to make a replicaof the master plate into a 96-well plate with the inducer and grow thatplate overnight.

An aliquot of the un-induced culture from each well was transferred intoa new 96-well plate containing 120 μl per well of selective media+0.2%arabinose. The cultures containing the inducer were grown overnight(e.g., 15-20 hours) at 37° C. without shaking to induce RT production.An aliquot (5 μl) of culture from each well was then transferred intoanother 96-well plate containing 5 μl per well of 2×PLD (50 mM Tris-HCl,pH 8.0, 20 mM EDTA, 1.8% (w/v) sucrose, 1% (v/v) Triton X-100, 10 mMDTT, and 2 mg/ml lysozyme) at room temperature. These extracts weresometimes assayed directly for reverse transcriptase before the heatingstep. The amount of RT activity in a 5-μl aliquot of extract was withinthe linear range of the assay. Lysates were stable at room temperaturefor at least 1 hr.

The extracts were heated, for example, using a water bath orthermocycler, for 5 or 10 minutes at temperatures that ranged from 50°C. to 60° C. Preferably, the cultures were heated for 5 minutes at 52°C. After heating and cooling to room temperature, RT activity in a 5-μlaliquot from the lysate in each well was assayed with (rC)_(n).(dG)₁₅ inanother 96-well plate. An aliquot (5 to 10 μl) of the extract was mixedwith 1.25×RT reaction mix. This reaction was placed in a 37° C. waterbath for 10 minutes. A small aliquot of the reaction mixture (5 μl) wasspotted onto a charged nylon membrane (Genescreen+, NEN). The membranewas washed twice with 10% TCA+1% sodium pyrophosphate, rinsed withethanol, dried, and placed next to a phosphor screen. As an alternative,the membrane may be washed twice with 4% sodium pyrophosphate (pH 8.0),rinsed with ethanol, dried, and then placed next to a phosphor screen.Radioactive product that had been trapped on the filter was detected byanalyzing the screen in a Posphorimager, using ImageQuant software(Molecular Devices).

Candidates were selected if they showed more reverse transcriptaseactivity (radioactivity) after the heat inactivation step. Thesecandidates were screened a second time to confirm the phenotype.Candidates which appeared to be thermostable after the second screenwere grown in small cultures and tested a third time for thermostablereverse transcriptase activity. Candidates that were reproducibly heatresistant were sequenced and the mutation in each clone was determined.

Plasmid DNA was prepared from an over night E. coli culture bearing anRT mutant using a Concert High Purity Miniprep Kit (InvitrogenCorporation, Carlsbad, Calif.) following the manufacturer'sinstructions. Each DNA was sequenced using a forward and reverse primerbordering the segment that had been mutagenized to generate the mutant.The sequencing reactions were carried out as specified for plasmid DNAusing the ABI Big Dye Terminator Sequencing Ready Reaction Kit. Thereactions were analyzed using an ABI PRISM 377 DNA Sequencer.

An oligonucleotide corresponding to the mutagenized site was designed inwhich the codon for the mutagenized amino acid was randomized (NNK orNNN). Oligonucleotide site-directed mutagenesis was carried out byestablished procedures. Saturation of an amino acid coding site in theH-RT gene with all possible amino acids was performed by introducing thesequence NNK (N=A,C,G or T and K=G or T) at the codon site into themutagenic oligonucleotide. These oligonucleotides were used insite-directed mutagenesis to generate a library in which all possiblesubstitutions at the mutagenized site were made. This library wasscreened for thermostable reverse transcriptase activity, and the mostpromising clones were sequenced.

Screening of mutants in Segment 2 (see FIG. 2) resulted in theidentification of one mutant, H204R. Screening of a library mutagenizedat site H204 resulted in several mutants, but the only one that was morethermostable than M-MLV reverse transcriptase was another H204R mutant.H204R mutants of M-MLV reverse transcriptase have enhancedthermostability. Screening of mutants in segment 3 (see FIG. 2) resultedin one mutant, T306K. Randomization of the T306 position producedthermostable mutants which, when sequenced, were T306R. Both T306K andT306R mutants of M-MLV reverse transcriptase have about 1.5 foldenhanced thermostability.

Example 3 TdT Reverse Transcriptase Mutants

In checking fidelity mutants of reverse transcriptase (RT) formisextension in a 3 dNTP assay, it was observed that SUPERSCRIPT™ IIreverse transcriptase extended 2-3 bases past the end of the template inthe presence of 3 and 4 dNTPs. This non-template directed extension orTdT activity is reduced in many mutants, but in a few such as F309N andT197E it appears that this activity is severely reduced or eliminated.These mutants are probably in close proximity or in contact with thetemplate-primer as determined by homology to HIV reverse transcriptaseand its crystal structure with bound template-primer.

Methods

Mutagenesis

For F309N:

Primers were designed corresponding to the mutant position F309 with thesilent insertion of a NgoMIV restriction site at amino acid positions310-311. The primers encoded a random NNK sequence for this positiongenerating a random library of F309 mutants, where N is any of the fourbases and K is T or G. The primers along with internal SUPERSCRIPT™ IIreverse transcriptase primers at an upstream SstI restriction site and adownstream SalI restriction site were used in a standard PCR reaction(10 ng SUPERSCRIPT™ II reverse transcriptase template, 2 μM of eachprimer, 48 μl SuperMix (Invitrogen Corporation (Carlsbad, Calif.)) for20 cycles of 94° C. 15 sec, 55° C. 15 sec, 72° C. 30 sec) to generatetwo PCR fragments. These were a 240 base pair SstI-NgoMIV fragment and a200 base pair NgoMIV-SalI fragment. The fragments were isolated anddigested and ligated together and then inserted into the originalSUPERSCRIPT™ II reverse transcriptase clone cut with SstI and SalI. Theresulting ligation product was transformed in Max Efficiency DH10B(Invitrogen Corporation (Carlsbad, Calif.)) competent cells to createthe library of mutants at site F309. This library was then platedovernight for selection.

For T197E and Y133A:

The mutants T197E and Y133A were made by oligo-directed mutagenesis asdescribed in Kunkel, T. A. et al. Methods Enzymol. 204:125 (1991).Briefly, the SUPERSCRiPT™ II reverse transcriptase gene was insertedinto pBADhisA (Invitrogen Corporation) vector and named pBAD-SSII. Thisplasmid was transformed into DH11S cells and the cells were infectedwith M13K07 helper phage from which single strand DNA was isolated.Oligos were designed corresponding to each mutation: T197E and Y133A.Each oligo (100 μM) was kinased with T4 polynucleotide kinase(Invitrogen Corporation (Carlsbad, Calif.)) using the Forward ReactionBuffer (Invitrogen Corporation (Carlsbad, Calif.)). The oligo wasannealed to single stranded pBAD-SSII DNA. Native T7 DNA polymerase(USB) and T4 DNA ligase (Invitrogen Corporation (Carlsbad, Calif.)) wereadded with synthesis buffer (0.4 mM dNTPs, 17.5 mM Tris-HCl, pH 7.5, 5mM MgCl₂, 2.5 mM DTT, and 1 mM ATP) to the annealed reaction on ice. Thereactions were incubated at 37° C. for 30 minutes and terminated byadding 1 μl of 0.5 M EDTA. The reactions were transformed and platedwith DH10B cells. Colonies were picked and mutants were determined byrestriction enzyme analysis and sequenced for confirmation using an ABI377 instrument and ABI Big Dye Terminator Cycle Sequencing ReadyReaction kit.

Selecting Colonies Containing Active Reverse Transcriptase.

Individual transformant colonies were inoculated into single wells of a96 well culture plate. Each well contained 120 μl of media (EG-Ap)containing 0.2% arabinose. It is preferable to first inoculate a 96 wellplate with selective medium without the inducer, to grow that masterplate overnight, and then to make a replica of the master plate into a96-well plate with the inducer and grow that plate overnight. Thecultures were grown overnight at 37° C. without shaking. Overnightcultures were mixed with an equal volume of 2×PLD (1.8% glucose, 50 mMTris-HCl, pH 8.0, 20 mM EDTA, 20 mM DTT, 1% Triton X-100, 2 mg/mLlysozyme) at room temperature. These extracts were assayed directly forreverse transcriptase activity by mixing 10 μl of the extract with 40 μlof 1.25×RT reaction mix (62.5 mM Tris-HCl, pH 8.4, 62.5 mM KCl, 12.5 mMMgCl₂, 12.5 mM DTT, 1.25 mM dGTP, polyC/oligo dG (3.75 mM/1.5 mM innucleotide), [³²P] dGTP). This reaction was placed in a 37° C. waterbath for 10 minutes. A small aliquot of the reaction mixture (5 μl) wasspotted onto a charged nylon membrane (Genescreen+, NEN). The membranewas washed twice with 10% TCA+1% sodium pyrophosphate, rinsed withethanol, dried, and placed next to a phosphor screen. Radioactiveproduct that had been trapped on the filter was detected by analyzingthe screen in a Phosphorimager, using ImageQuant software (MolecularDevices). Candidates were selected if they showed reverse transcriptaseactivity (radioactivity). These candidates were screened a second timeto confirm the phenotype. The confirmed candidates were then sequencedto determine which amino acids maintained detectable reversetranscriptase activity.

Purification of Reverse Transcriptase Mutants.

The cell pellet containing induced reverse transcriptase was suspendedin a ratio of 2 mL Lysis buffer (40 mM Tris-HCl, pH 8.0, 0.1 M KCl, 1 mMPMSF)/1 gram of cell pellet. The suspension was sonicated on ice andthen centrifuged at 27,000 g for 30 minutes. The cell-free extract wasfiltered through a 0.45μ syringe filter. The cell-free extract wasapplied to a 5 mL Ni²⁺ HI-TRAP column (Pharmacia) pre-equilibrated with5 volumes 5 mM imidazole in buffer A (40 mM Tris HCl, pH 8.0, 10%glycerol, 0.01% Triton X-100, 0.1 M KCl) at 1 mL/min. The column waswashed with 10 volumes 5 mM imidazole in buffer A. The reversetranscriptase was eluted by washing with 20 volumes of a gradient of 5mM to 1M imidazole in buffer A. The eluate containing reversetranscriptase protein was applied to a 1 mL Mono-S column (Pharmacia)pre-equilibrated with 10 column volumes 50 mM KCl in buffer B (40 mMTris-HCl, pH 8.0, 10% glycerol, 0.01% Triton X-100, 0.1 mM EDTA, 1 mMDTT) at a flow rate of 1.0 mL/min. The column was washed with 10 volumesof 50 mM KCl in buffer B. Reverse transcriptase was eluted with 20volumes of a gradient from 50 mM to 1 M KCl in buffer B. The individualfractions were analyzed for RT activity. The fraction containing peak RTactivity was dialyzed against storage buffer (40 mM Tris-HCl, pH 8.0,50% glycerol, 0.01% Triton X-100, 0.1 mM EDTA, 1 mM DTT, 0.1 M KCl). Thepurified reverse transcriptases were more than 95% pure, as judged bySDS-PAGE. The protein concentrations were determined by using the Bioradcolorimetric kit.

3 dNTP Assay Method.

Procedures were modified from those of Preston, B. D., et al. Science242:1168 (1988). The DNA template-primer was prepared by annealing a47-mer template (5′-GAGTTACAGTGTTTTTGTTCCAGTCTGTAGCAGTGTGTGAATGGAA G-3′)(SEQ ID NO:6) to an 18-mer primer (5′-CTTCCATTCACACACTGC-3′) (SEQ IDNO:7) [³²P]-labeled at the 5′-end with T4 polynucleotide kinase(template:primer, 3:1). Assay mixture (10 μl) contained 5 nMtemplate-primer, 50-200 nM reverse transcriptase as specified in figurelegends, 3 or 4 dNTPs (250 μM each), 50 mM Tris-HCl (pH 8.3), 75 mM KCl,3 mM MgCl₂, 10 mM DTT. Reactions were incubated at 37° C. for 30 minutesand terminated by the addition of 5 μl of 40 mM EDTA, 99% formamide.Reaction products were denatured by incubating at 95° C. for 5 minutesand analyzed by electrophoresis on urea 6% polyacrylamide gels.

To determine if any TdT activity was occurring in the control reactionof the 3 dNTP assay, which uses all 4 dNTPs, the control reaction wasrepeated with varying amounts of enzyme, >600 units to 20 units, at 37°C. for 30 minutes. For SUPERSCRIPT™ II, T197E, and Y133A, 200, 100, 50,and 20 units were used. For F309N, 646, 200, 50, and 20 units were used.

Results

We carried out a misinsertion assay of F309N(H204R, T306K) SUPERSCRIPT™II reverse transcriptase, hereafter referred to as F309N, with DNAtemplate. This assay was employed to compare the misincorporationcapability of the mutant to SUPERSCRIPT™ II. The assay is a primerextension assay using synthetic DNA template-primer and biased dNTPpools containing only three of four dNTPs. The reactions are displayedon a gel in FIG. 3. While conducting this procedure to screen formutants with lower misinsertion/misextension rates it was observed thatSUPERSCRIPT™ II reverse transcriptase extended 2-3 nucleotides past thetemplate end and that some mutations reduced or appeared to eliminatethis non-template directed extension or TdT activity. As shown in FIG.4, in the presence of all 4 dNTPs, SUPERSCRIPT™ II reverse transcriptaseand the mutant F309N were able to extend the primer approximatelyequally, with SUPERSCRIPT™ II reverse transcriptase adding 2 nucleotidespast the template, and F309N adding none beyond the end of the template.To further evaluate this non-templated directed extension the controlreaction for the 3 dNTP misextension assay containing all 4 dNTPs wascarried out with SUPERSCRIPT™ II, F309N, T197E, and Y133A reversetranscriptase for 30 minutes with varying amounts of enzyme. The threemutants had shown very reduced levels of TdT activity in prior screens.Since it had been observed that 5 minutes with 20 units of enzyme wasmore than enough time for the primer extension to be completed, a 30minute incubation and 200 to 646 units of reverse transcriptase wereboth in large excess over what was necessary for the reaction to becompleted. As seen in FIG. 4, all the reverse transcriptase reactions atthe lowest amount tested had similar extension products to the reactionsat the highest unit concentrations demonstrating that the reaction hadgone to completion. SUPERSCRIPT™ II reverse transcriptase added 2nucleotides past the end of the template, F309N and T197E did not extendpast the end of the template, and Y133A appears to have a small amountof product that is 1 nucleotide past the end of the template.

Example 4 Dual Thermostable and TdT Mutants

The F309 amino acid position in M-MLV reverse transcriptase (RT) alignswith the W266 position in HIV reverse transcriptase. This position is atthe base of the thumb domain and is considered part of the minor groovebinding tract which interacts with the minor groove of thetemplate-primer. The mutations H204R and T306K have been shown toincrease the thermostability of the enzyme. The F309N mutation in anH204R/T306K clone displays 2.3× lower mutation frequency in a lacZforward assay (Table 5) on RNA template and shorter extension productsin a 3 dNTP extension assay than SUPERSCRIPT™ II reverse transcriptaseor H204R/T306K in SUPERSCRIPT™ II reverse transcriptase. Both findingssupport the claim of an enzyme with higher fidelity (Table 6).

TABLE 5 Mutation Frequency of M-MLV Reverse Transcriptase High FidelityMutants total mutant MF Construct plaques plaques (×10⁻⁴) SUPERSCRIPT ™II 15689 87 39 SUPERSCRIPT ™ II 14410 83 41 (H204R, T306K) SUPERSCRIPT ™II 11623 39 17 (H204R, T306K, F309N) SUPERSCRIPT ™ II 11415 39 14(H204R, T306K, F309N, V223H)Table 5. The mutation frequency of SUPERSCRIPT™ II reverse transcriptaseand point mutants. Mutation frequency (MF) was determined by dividingthe number of mutant plaques (light blue or white) by the total numberof plaques. The background mutant frequency of the starting DNA was17×10⁻⁴ for the first 3 constructs and 20×10⁻⁴ for the last construct.

TABLE 6 Error Rates of M-MLV Reverse Transcriptase High Fidelity MutantsV223H/ M-MLV SUPERSCRIPT ™ II F309N F309N Overall ER 1/17,000 1/15,0001/34,000 1/41,000 (oER) Mismatch % of total 46 35 68 72 ER (mER)1/37,000 1/42,000 1/50,000 1/58,000 Frameshift % of total 46 60 21 22 ER(rER) 1/37,000 1/25,000 1/162,000 1/188,000 Strand Jump % of total  8  511 6 ER (jER) 1/213,000 1/297,000 1/324,000 1/690,000

Methods

Mutagenesis. Using a standard site directed mutagenesis protocol, asdescribed in Example 3, a primer containing the V223H mutation wasannealed to single strand DNA of SUPERSCRIPT™ II with the followingmutations: H204R, T306K, F309N. The colonies were sequenced to confirmthe new combination of V223H, H204R, T306K, and F309N.

Selecting Colonies Containing Active Reverse Transcriptase. Colonyselection was performed as in Example 3.

Purification of RT mutants. Purification was performed as in Example 3.

Sequencing of plaques. The plaques from the lacZ forward assay weretransferred from the soft agar plate to Whatmann 3MM paper and allowedto dry for at least 1 hour. The plaque was then punched out and theplaque/paper disk was added directly to a sequencing reaction mixcontaining 4-8 μl ABI PRISM Dye Terminator Cycle Sequencing ReadyReaction (Perkin Elmer), 1 μl primer (GAAGATCGCACTCCAGCCAGC) (SEQ IDNO:8), and distilled water to 20 μl total volume. The ABI cyclesequencing protocol was used for 96° C. 10 seconds, 50° C. 5 seconds,60° C. 4 minutes for 25 cycles. The paper disks were removed and thereactions were precipitated, then resuspended in loading dye and run onan ABI 377 sequencing machine.

The sequences were compared to wild type lacZ alpha sequence and thenclassified as frameshift (either 1 nucleotide insertion or deletion),mismatch, or strand jump (an insertion or deletion between repeatedsequences). The overall error rate for each class was determined bydividing the mutation frequency by the number of detectable sites (i.e.,sites the alteration of which results in a phenotypic change) (116)multiplied by 0.5 (to exclude the original single strand contribution)and then multiplied by the percentage of mutants observed to be in eachclass. ER=MF/(detectable sites*0.5)*(% in each class).

3dNTP assay method. 3dNTP assays were performed as in Example 3.

Results

We carried out a misinsertion assay of F309N(H204R T306K) SUPERSCRIPT™II reverse transcriptase, hereafter referred to as F309N, and V223HF309N(H204R T306K), hereafter referred to as V223H/F309N with DNAtemplate. This assay was employed to compare the misincorporationcapability of the mutant to SUPERSCRIPT™ II. The assay is a primerextension assay using synthetic DNA template-primer and biased dNTPpools containing only three of the four dNTPs. The reactions aredisplayed on a gel in FIG. 5 and FIG. 6. In this assay, higherefficiency of primer extension denotes lower fidelity. As shown in FIGS.5 and 6, in the presence of all 4 dNTPs, SUPERSCRIPT™ II reversetranscriptase and the mutants F309N and V223H/F309N were able to extendthe primer approximately equally, with some variance in the addition ofnon-template directed nucleotides at the end of the primer. However whenincubated with biased pools of nucleotides, SUPERSCRIPT™ II reversetranscriptase was able to catalyze substantial extension past templatenucleotides for which a complementary dNTP was missing, indicating useof incorrect nucleotides and lower fidelity. In FIG. 5, the F309N (2)mutant showed shorter extension products than SUPERSCRIPT™ II reversetranscriptase in each of the biased pools of three dNTPs, indicatingless ability to incorporate incorrect nucleotides and thus higherfidelity. In FIG. 6, the V223H/F309N mutant was extended with just thedATP and dCTP pools. In each case V223H/F309N also had lower extensionproducts than SUPERSCRIPT™ II. This corresponds with the results of thelacZα assay where the F309N and V223H/F309N mutants had a lower mutationfrequency than SUPERSCRIPT™ II reverse transcriptase (17×10⁻⁴ and14×10⁻⁴ to 39×10⁻⁴). The reverse transcriptase with just the H204R T306Kmutations without F309N has a mutation frequency similar to SUPERSCRIPT™II reverse transcriptase (41×10⁻⁴ to 39×10⁻⁴), suggesting that thesemutations do not influence fidelity. This data shows a correlationbetween the misinsertion assay on DNA and the lacZα assay on RNA whereinhigher fidelity mutants had both shorter extension products with biasedpools of dNTPs and lower mutation frequencies in the lacZα assay.

Example 5 Error Rate Determination

To determine Error Rates, mutant plaques from the lacZ forward assaywere sequenced using known methods. The mutations were then classifiedinto one of the following categories: mismatches for misinsertionevents, frameshifts for single insertion or deletion events, or jumpsfor large insertions or deletions caused by jumping between similarsequences. An overall Error Rate was then determined for nucleic acidencoding the lacZ alpha peptide using the following equation:ER(error rate)=MF(mutation frequency)/(number of detectable sites×0.5),where the number of detectable sites is 116.

Not all bases mutated in lacZ forward assays result in a detectablephenotypic change. To determine specific error rates for mismatch,frameshift and jumps, the mutation frequency was modified by multiplyingby the percent of the total of each mutant category, and then used todetermine the specific error rate. The following is a sequence map ofthe lacZα peptide in M13 mp19 from SUPERSCRIPT™ II reverse transcriptaseand the high fidelity SUPERSCRIPT™ II H203R T306K F309N reversetranscriptase assays. Underlining indicates deletions; “^” indicatesinsertions of the base A, T, C, or G shown above; A, T, C, or G shownabove the complete sequence indicates mismatches.

Map of Mutations Introduced by SUPERSCRIPT ™ II                                                     T C       T                      T                     TC CAGCGCAACGC AATTAATGTG AGTTAGCTCA CTCATTAGGC ACCCCAGGCT TTACACTTTA                  1                  1               4              CG                                  C      CCTGCTTCCGGC TCGTATGTTG TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA      1    C     CC                CG       C GCTATG ACC ATG ATT ACG{circumflexover ( )}CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG                                                   1                                       T                          AAAA                                       T A                        AAA                     T                 T A                         A                     T       T         T A               T         A    CGTA CCG AGC TCG AAT TCA CTG GCC GTC GTT{circumflex over( )}TTA CAA CGT CGT GAC TGG GAA AAC CCT GGC                                     7                       1   1      1                                               TTTTT                                               TTTTT                                           C   TTTTT                                           C   TTT                                           A T T        

    

           

        

  

   

  

 

 

    

    

     

GTT ACC CAA CTT AAT CGC CTT GCA GCA CAT CCC{circumflex over( )}CCT{circumflex over ( )}TTC{circumflex over ( )}GCC AGC TGG CGT                                        1     4

 (SEQ ID NO: 9)

TABLE 7 Insertions 40 38% 60% frameshift (insertion or deletion)Deletions 23 22% Mismatches 36 35% 35% mismatch Jumps 5  5%  5% Jumps

TABLE 8 Overall Error Rate (oER) 1/15,000 (39 × 10⁻⁴)/(116 × 0.5)Mismatch Error Rate 1/42,500 (0.35 × 39 × 10⁻⁴)/(116 × 0.5) (mER)Frameshift Error Rate (fER) 1/25,000 (0.60 × 39 × 10⁻⁴)/(116 × 0.5)Jumps Error Rate (jER) 1/297,000 (0.05 × 39 × 10⁻⁴)/(116 × 0.5)

Example 6 Analysis of Enzymatic Activity in Mutant RTs

Reverse transcriptase (RT) (e.g., retroviral RT) is one of the mostintensely studied DNA polymerases, both because of its use as anessential tool for the synthesis and cloning of cDNA and because of itsimportance as a target for inhibition of HIV (Skalka, A. M. and Goff, S.P., Reverse Transcriptase, Cold Spring Harbor Laboratory Press, ColdSpring, N.Y. (1993)). A dichotomy exists however in that HIV RT, thefocus of much of recent study (Le Grice, S. F. J. in ReverseTranscriptase (Skalka, A. M. and Goff, S. P., eds) Cold Spring HarborLaboratory Press, Cold Spring, N.Y., pp. 163-191, (1993)), has not beenused widely as a tool for cDNA synthesis. This is because HIV RT has arelatively high error rate (Bebenek, K. and Kunkel, T. A. in ReverseTranscriptase (Skalka, A. M. and Goff, S. P., eds) Cold Spring HarborLaboratory Press, Cold Spring, N.Y., pp. 85-102 (1993), and because itdoes not synthesize efficiently full-length copies of long mRNAs invitro. Other forms of retroviral RT used widely to synthesize cDNA,M-MLV RT and AMV RT, also suffer from these limitations, but to a lesserextent (Bebenek, K. and Kunkel, T. A. in Reverse Transcriptase (Skalka,A. M. and Goff, S. P., eds) Cold Spring Harbor Laboratory Press, ColdSpring, N.Y., pp. 85-102 (1993); Krug, M. S. and Berger, S. L. Methodsin Enzymol. 152:316-325 (1987); Gerard, G. F. and D'Alessio, J. M.(1993) in Methods in Molecular Biology, Vol 16: Enzymes of MolecularBiology (Burrell, M. M., ed) pp. 73-93, Humana Press, Totowa, N.J.(1993); Gerard, G. F., et al., Molecular Biotechnology 8:61-77 (1997)).In addition to polymerase activity, retroviral RT possesses RNase Hactivity that degrades the RNA in an RNA-DNA hybrid (Moelling, K., etal., Nature New Biology 234:240-244 (1971)). The presence of thisdegradative activity is responsible in part for the limitation onefficient synthesis of long cDNA (Krug, M. S. and Berger, S. L. Methodsin Enzymol. 152:316-325 (1987), Berger, S. L., et al., Biochem.22:2365-2372 (1983)). The RNase H domain of RT can be mutated to reduceor eliminate RNase H activity while maintaining mRNA-directed DNApolymerase activity (Kotewicz, M. L., et al., Nuc. Acids Res. 16:265-277(1988), DeStefano, J. J., et al., Biochim. Biophys. Acta 1219:380-388(1994)), improving the efficiency of cDNA synthesis (Kotewicz, M. L., etal., Nuc. Acids Res. 16:265-277 (1988)).

A second significant drawback to copying mRNA is the tendency of RT topause during cDNA synthesis resulting in the generation of truncatedproducts (Harrison, G. P., et al., Nuc. Acids Res. 26:3433-3442 (1998),DeStefano, J. J., et al., J. Biol. Chem. 266:7423-7431 (1991)). Thispausing is due in part to the secondary structure of RNA (Harrison, G.P., et al., Nuc. Acids Res. 26:3433-3442 (1998), Wu, W., et al., J.Virol. 70:7132-7142 (1996)). Performing cDNA synthesis at reactiontemperatures that melt the secondary structure of mRNA helps toalleviate this problem (Myers, T. W. and Gelfand, D. H., Biochem.30:7661-7666 (1991)). In addition, the oligo(dT)_(n) primer often usedto initiate cDNA synthesis tends to prime at internal stretches of Aresidues in mRNA at lower temperatures, resulting in the synthesis of3′-end truncated cDNA products. M-MLV RT does not efficiently synthesizecDNA from mRNA above 43° C. (Tosh, C., et al., Acta Virol. 41:153-155(1997)). RNase H-minus (H−) M-MLV RT can be used up to 48° C. because inthe absence of RNase H activity the mRNA template-DNA product complex ismaintained during cDNA synthesis in a structural form that protects RTfrom thermal inactivation.

In an effort to raise the temperature at which M-MLV RT can be used tosynthesize cDNA, we have randomly mutagenized the H− M-MLV RT gene andscreened for thermal stable mutants. Several thermal stable mutants ofH− M-MLV RT were identified and purified enzymes were characterized. Weshow that when the mutations are present together they increase RTthermal activity by increasing its intrinsic thermal stability withoutaltering catalytic activity.

Experimental Procedures

Bacterial Strains and Plasmids—E. coli DH10B (Invitrogen) was used inall experiments. Bacterial liquid cultures were grown at 37° C. in EG:2% tryptone, 1% yeast extract, 0.5% glycerol, 10 mM NaCl, and 1 mM KCl.Solid medium was LB (1% bactotryptone, 0.5% yeast extract, and 86 mMNaCl)+1.5% agar. Selective media included 100 μg/ml ampicillin. Forinduction of cultures, cells were inoculated into selective EG+0.2%arabinose and grown for 18 hr.

RNA and DNA—Chloramphenicol acetyl transferase (CAT) cRNA (˜900 nt) witha 40-nucleotide poly(A) tail at the 3′-end was synthesized by T7 RNApolymerase run-off transcription from linearized plasmid DNA (D'Alessio,J. M. and Gerard, G. F., Nuc. Acids Res. 16:1999-2014 (1988)). The cRNAwas selected on oligo(dT)-cellulose to ensure the presence of a poly(A)tail. For labeling, the 5′ end of CAT cRNA was dephosphorylated withalkaline phosphatase. (rC)_(n), p(dT)₁₂₋₁₈, and p(dT)₂₅₋₃₀ werepurchased from Amersham Pharmacia. (rA)₆₃₀ was purchased from Miles.(dG)₁₅ and (dT)₂₀ were from Invitrogen. A DNA 24-mer complementary toCAT cRNA that annealed between nucleotides 679 and 692 with its 5′ end146 nucleotides distant from the first base at the 5′ end of the CATcRNA poly(A) tail was from Invitrogen. Oligonucleotides to prime PCR andperform site-directed mutagenesis were from Invitrogen.

M-MLV RT Gene—The M-MLV RT gene used in these studies was derived frompRT601 (Kotewicz, M. L., et al., Nuc. Acids Res. 16:265-277 (1988),Gerard, G. F., et al., DNA 5:271-279 (1986)). The gene starting atnucleotide 2598 and ending at nucleotide 4628 (Shinnick, T. M., et al.,Nature 293:543-548 (1981)) was cloned under control of an araD promoterinto plasmid pBAD/HisA (Invitrogen). The M-MLV gene was further modifiedby site-directed mutagenesis without changing amino acid coding toinclude several unique restriction endonuclease sites that divided thegene into five segments (FIG. 2A). The amino end of the proteincontained a His₆ tag to simplify purification that included thefollowing amino acids: MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH (amino acids1-32 of SEQ ID NO:2). The carboxy end of the protein contained theadditional amino acids NSRLIN present as the result of subcloning frompRT601 (17). In addition, the M-MLV RT gene was mutated (D524G, E562Q,D583N) to eliminate RNase H activity. The final construct was designatedpBAD-HSS2 (FIG. 2B), and the gene and gene product were designated His₆H-RT.

DNA Sequencing—Plasmid DNA was prepared from an over night E. coliculture bearing an RT mutant using a Concert High Purity Miniprep Kit(Invitrogen) following the manufacturer's instructions. Each DNA wassequenced using a forward and reverse primer bordering the segment thathad been mutagenized to generate the mutant. The sequencing reactionswere carried out as specified for plasmid DNA using the ABI Big DyeTerminator Sequencing Ready Reaction Kit. The reactions were analyzedusing an ABI PRISM 377 DNA Sequencer.

Random Mutagenesis—Mutagenic PCR was performed in a reaction mixture(100 μl) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl₂,0.3 mM MnSO₄, 200 μM each of dCTP, dGTP, dTTP, and dATP, and 0.5 unitsof Taq DNA polymerase (Invitrogen Corporation, Carlsbad, Calif.). Aftera 1 min denaturation step at 94° C., the cycling protocol was 15 sec at94° C., 15 sec at 55° C., and 30 sec at 72° C. for 20 cycles.Amplification was 100 fold from 50 ng of target to 5 μg of amplifiedproduct. PCR primers included appropriate restriction endonuclease cutsites. An amplified DNA segment was cleaved with appropriate restrictionendonucleases, gel purified, and cloned into gel purified vector DNA cutwith the corresponding restriction enzymes.

Site-directed Mutagenesis—Oligonucleotide site-directed mutagenesis wascarried out by established procedures (Kunkel, T. A., et al., MethodsEnzymol. 154:812-819 (1987), Kunkel, T. A., et al., Methods Enzymol.204:125-139 (1991)). Saturation of an amino acid coding site in the H-RTgene with all possible amino acids was performed by introducing thesequence NNK (N=A,C,G or T and K=G or T) at the codon site into themutagenic oligonucleotide.

DNA Polymerase Assays—During screening of RT mutants, RT RNA-directedDNA polymerase activity was assayed with (rC)_(n).(dG)₁₅, which isspecific for RT under the reaction conditions used (Gerard, G. F., etal., Biochem. 13:1632-1641 (1974)). Reaction mixtures (50 μl) containing50 mM Tris-HCl (pH 8.4), 50 mM KCl, 10 mM MgCl₂, 300 μM (rC)_(n), 120 μM(dG)₁₅, 0.01% (v/v) Triton X-100, and 100 μM [α-³²P]dGTP (1,000cpm/pmole), were incubated at 37° C. for 10 min in the wells of a96-well plate. An aliquot (5 μl) from each well was spotted onto aGenescreen+(NEN) filter and the filter was washed twice for 10 min with4% (w/v) sodium pyrophosphate (pH 8.0). Radioactivity bound to the driedfilter was quantified in a phosphorimager (Molecular Dynamics).

RT DNA polymerase unit activity was assayed with (rA)₆₃₀.p(dT)₁₂₋₁₈(Houts, G. E., et al., J. Virol. 29:517-522 (1979)). One unit of DNApolymerase activity is the amount of RT that incorporates one nmole ofdeoxynucleoside triphosphate into acid insoluble product at 37° C. in 10min.

cDNA synthesis from CAT cRNA was carried out in reaction mixtures (20p. 1) containing 50 mM Tris-HCl (pH 8.4), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiotreitol (DTT), 500 μM each of dATP, dTTP, dGTP, and [α-³²P]dCTP(300 cpm/pmole), 1,750 units/ml RNase Inhibitor, 130 μg/ml (465 nM) CATcRNA, 20 μg/ml (2,300 nM) p(dT)₂₅₋₃₀, and 3,250 units/ml (100 nM) RT.Incubation was at various temperatures for 60, min in individual tubes.An aliquot of the reaction mixture was precipitated with TCA todetermine yield of cDNA synthesized, and the remaining cDNA product wassize fractionated on an alkaline 1.2% agarose gel (McDonell, M. W., etal., J. Mol. Biol. 110:199-146 (1977)).

To establish mono- and divalent metal reaction optima, initial reactionrates were determined under conditions of limiting RT concentrationduring a 10-min incubation at 37 or 50° C. Reaction mixtures (20 μl)contained 50 mM Tris-HCl (pH 8.4), 10 mM DTT, 500 μM each of dTTP, dATP,dCTP, and [³H]dGTP (100 cpm/pmole), 10 pmoles (2.8 μg) CAT cRNA, 50pmoles DNA 24-mer, 0.5 pmoles RT, and KCl and MgCl₂, varied inconcentration one at a time.

Steady-state Kinetic Measurements—The steady-state kinetic parametersK_(m(dTTP)) and k_(cat) were determined as described (Polesky, A. H., etal., J. Biol. Chem. 265:14579-14591 (1990)), using (A)_(n).(dT)₃₀. Arange of five [³²P]dTTP concentrations, which bracketed the K_(m(dTTP))value, was used for each determination of the kinetic parameters. Theconcentration of the template-primer and enzyme in the reaction were 2μM (in primer termini) and 4 μM, respectively. Reaction mixtures (50 μl)also contained 50 mM Tris-HCl, pH 8.4, 75 mM KCl, 3 mM MgCl₂, and 10 mMDTT and were incubated at 37° C.

Mutant Screening—Mutant populations were plated on selective agar.Individual colonies were inoculated into 120 μl of selective EG in 96well plates and grown overnight at 37° C. The cell density of thecultures was ˜10⁹ cfu/ml. An aliquot of the culture from each well wastransferred into a new 96-well plate containing 120 μl per well ofselective media+0.2% arabinose. This plate was incubated at 37° C. for20 hr to induce expression of RT. An aliquot (5 μl) of culture from eachwell was then transferred into another 96-well plate containing 5 μl perwell of 2×PLD (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.8% (w/v) sucrose,1% (v/v) Triton X-100, 10 mM DTT, and 2 mg/ml lysozyme). After heatingfor 5 to 10 min at various temperatures (52 to 58° C.) in a thermocyclerand cooling to room temperature, RT activity in a 5-μl aliquot from thelysate in each well was assayed with (rC)_(n).(dG)₁₅ in another 96-wellplate as described above. The amount of RT activity in a 5-μl aliquot ofextract was within the linear range of the assay. Lysates were stable atroom temperature for at least 1 hr.

Purification of RTs—All operations were at 4° C. Induced E. coli cells(5 g) bearing pBAD-HSS2 or a derivative were suspended in 10 mL ofbuffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl, and 1 mM PMSF) and weresonicated for disruption. After clarification by centrifugation at20,000×g for 30 min, RT was purified by sequential chromatography on a5-ml Chelating Sepharose column charged with Ni²⁺ and a Mono S HR 5/5column (Amersham Pharmacia). In some cases the RT was fractionated on athird column (AF-Heparin-650 from TosoHaas) to eliminate traces of RNasecontamination. The purified RT was dialyzed against storage buffer (40mM Tris-HCl, pH 8.0, 100 mM KCl, 0.01% (v/v) Triton X-100, 0.1 mM EDTA,1 mM DTT, and 50% v/v glycerol) and stored at −20° C. RT purified bythis procedure was >90% homogeneous as judged by SDS PAGE and was freeof detectable contaminating DNA endonuclease, DNA exonuclease, and RNAendonuclease.

Thermal Inactivation Profile of RT in Extracts—Cell lysates prepared inPLD as described already were heated in a 96-well plate in athermocycler in which a temperature gradient exists through the rows,but the temperature in each column is the same. After incubation for 5min at temperatures ranging from 25° C. to 56° C., RT activity wasassayed with (rC)_(n).(dG)₁₅ as described already.

Half Life Determination—Mixtures (20 μl) were incubated for varioustimes in 0.5-ml tubes in a thermocycler at 50 or 55° C. and contained 50mM Tris-HCl (pH 8.4), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 0.1% (v/v)Triton X-100, and 3-7 μg/ml purified RT. Incubation was stopped byplacing the tube in ice. An aliquot (5 μl) was assayed for residualactivity with (rA)₆₃₀.p(dT)₁₂₋₁₈.

Measurement of K_(D) by Filter Binding—A nitrocellulose filter-bindingassay (Bailey, J. M., Anal. Biochem. 93:204-206 (1979), Strauss, H. S.,et al., Gene 13:75-87 (1981)) was used to determine the nucleic acidbinding constants (K_(D)) of RTs for CAT cRNA.(dT)₂₀. DephosphorylatedCAT cRNA was labeled at the 5′ end with [γ-³²P]ATP and T4 polynucleotidekinase (Boehringer). Oligo(dT)₂₀ was annealed to the poly(A)-tailed CATcRNA in a buffer containing 10 mM Tris-HCl, pH 7.5, and 80 mM KCl at 65°C. for 5 min followed by chilling on ice. Reaction mixtures (100 μl)containing binding buffer (50 mM Tris-HCl, pH 8.4, 75 mM KCl, 3 mMMgCl₂, and 10 mM DTT), 0.05 nM ³²P-labeled CAT cRNA, 1 nM (dT)₂₀, and 1to 50 nM RT were incubated at 23° C. for 5 min. After incubation, themixture was filtered through a nitrocelullose filter (Millipore, HA 0.45mm) soaked in binding buffer, which was then washed with binding buffer.The K_(D) is equal to that enzyme concentration at which 50% of thelabeled CAT cRNA is bound. For this method of analysis to be valid, theCAT cRNA concentration in the reaction must be substantially belowK_(D), so that the total enzyme concentration approximates theconcentration of free unbound enzyme.

Results

Mutants Generated by Random Mutagenesis—PCR primers were designed toamplify each of five segments of the M-MLV His₆ H− RT gene (FIG. 2A) andto contain appropriate restriction endonuclease cut sites. Each segmentwas randomly mutagenized by PCR mutagenesis (Experimental Procedures),digested with the appropriate restriction endonucleases, and cloned intoa similarly digested pBAD-HSS2 plasmid in such a way as to replace thecorresponding segment in the H− RT gene with the mutagenized segment(FIG. 2B). Libraries were sampled by DNA sequencing of the mutagenizedH− RT gene segment of a small number of clones to determine the mutationfrequency. The goal was to achieve rates of PCR random mutagenesis thatproduced 1 to 2 mutations per segment. We found that random mutagenesisthat produced greater than 2 mutations per segment tended to produce alarge proportion of inactive RT mutants. If the library met thiscriterion, further screening by heat treatment was carried out toidentify mutants that showed greater RT activity in lysates than H− RTafter a heat treatment step (Experimental Procedures; FIG. 7A). Thosemutants with the highest apparent thermal stability were screened againin duplicate by pretreatment at 24° C. (to normalize for activity) andat 52 to 58° C. to confirm the presence of enhanced thermal stability(FIG. 7B). In all, about 15,000 clones were screened by heat treatmentin the 96-well format for each segment or a total of about 100,000mutants.

Two mutants were found that produced RT with greater thermal stabilitythan H− RT. DNA sequencing of their genes revealed them to be H204R insegment 2 and T306R in segment 3. Having identified two amino acidlocations that influence RT thermal stability, we wished to establish ifthe particular mutant amino acid selected gave the greatest possibleincrease in thermal stability. The two sites in the H− RT gene wereindependently subjected to site-directed mutagenesis in which thesequence NNK was substituted for the existing codon in the mutagenicoligonucleotide (Experimental Procedures). Eighty-four members from eachof these libraries were screened for thermal stable mutants. The resultsshowed that T306K and T306R were the most thermal stable variants atT306, with T306K being somewhat more thermal stable, and H204R was themost thermal stable variant at H204.

The mutations were combined in one clone by sequential site-directedmutagenesis. Assay of heat-treated extracts showed that the doublemutant His₆ H− H204R T306K RT was more thermal stable than either mutantalone and much more thermal stable than His₆ H− RT (FIG. 8).

Utilizing plasmid pBAD-HSS2 that contained the H204R and T306K mutationsas a starting point, RT gene segments 1 through 5 were again randomlymutagenized and libraries were screened for additional mutations thatrendered His₆ H− H204R T306K RT more thermal stable. One additionalmutation, M289L, in segment 3 was identified that increased the thermalstability of the RT double mutant in crude extracts.

Mutants from Single-Site Mutagenesis—His₆ H− T306K RT was mutated atposition F309 to F309N by site-directed mutagenesis as part of a studyof the fidelity of H− RT. The F309N mutation increased the thermalstability of His₆ H− T306K RT in crude extracts.

Thermal Stability of Purified Mutant RTs—A number of RT mutants werepurified to near homogeneity (Experimental Procedures) in order toestablish their intrinsic thermal stability in the absence ofcontaminants and to characterize their enzymatic properties. Theintrinsic thermal stability of purified forms of single mutants of His₆H− RT at each of the 4 positions were established and compared with thatof the starting enzyme. Mutations T306K, M289L, or F309N increased thehalf-life of His₆ H− RT a small amount at 50° C. from 8 min to between10 and 13 min (Table 9).

TABLE 9 Half-lives of purified RT mutants at 50° C. Half-life at 50° C.Enzyme (min)^(a) H-RT (SUPERSCRIPT ™ II)  3.2 ± 0.2 His₆ H-RT 8 ± 1.2(1.6 ± 0.3)^(b) His₆ H-H204R RT 43 ± 9 His₆ H-T306K RT   10 ± 0.4 His₆H-F309N RT 13 ± 3 His₆ H-M289L RT 13 ± 1 His₆ H-H204R T306K RT 78 ± 8H-H204R T306K F309N RT 30 His₆ H-H204R T306K F309N RT 105 ± 11 His₆H-H204R M289L T306K 240 ± 10 (8.1 ± 0.3) F309N RT ^(a)Mean ± standarddeviation of two or three determinations ^(b)Half-lives at 55° C. are inparentheses

The single mutation with the greatest impact was H204R, which increasedthe half-life of RT at 50° C. 5-fold (Table 9). However, combining themutations had at a minimum a positive additive effect upon thermalstability. Combining H204R and T306K increased the half-life at 50° C.10-fold, and combining M289L and F309N with these two mutationsincreased the half-life at 50° C. another 3-fold to 240 min (a 30-foldincrease relative to the half-life of the starting enzyme; Table 9).

The half-lives of His₆ H− RT and all its mutants dropped off at 55° C.relative to 50° C. (Table 9). The magnitude of the relative increase inintrinsic half-life observed at 50° C. for the quadruple mutant wasmaintained partially at 55° C. (Table 9). The half-life of His₆ H− RT of1.6 min at 55° C. was increased 5-fold by H204R M289L T306K F309N to 8.1min (Table 9).

The presence of an N-terminal tag was seen to increase thermal stabilityof the RTs examined. A comparison of an RT with no N-terminal tag(SUPERSCRIPT™ II) with an RT having the same point mutations and havingan N-terminal tag with the sequence MGGSHHHHHHRHHGMASMTGGQQMGRDLYDDDDKHcorresponding to amino acids 1-32 of SEQ ID NO:2 (His₆ H− RT) shows thatthe presence of the tag increases the thermal stability of the RT. Thepresence of the tag increased the half-life of the RT at 50° C. by afactor of 2.5-fold (from 3.2 minutes to 8 minutes). A comparison of thetriple mutant H204R T306K F309N with and without tag showed an increasein the half-life of the enzyme at 50° C. by a factor of 3.5-fold (30minutes to 105 minutes). Thus, the present invention contemplates RTswith an increased thermal stability that comprise one or more aminoacids added to the N-terminal of the RT. In addition, the inventioncontemplates RTs with enhanced thermal stability that comprise one ormore amino acids added to the C-terminal of the RT.

Enzymatic Characterization of Purified Mutant RTs—A number of catalyticproperties of the purified RT mutants were compared to ascertain ifintroduction of all four point mutations in RT altered its DNA syntheticactivity. With one exception, all of the thermal stable mutantscharacterized had RNA-directed DNA polymerase specific activitiesgreater than that of starting His₆ H− RT, but within a factor of1.5-fold (Table 10).

TABLE 10 RNA-directed DNA polymerase specific activity of purified RTmutants DNA Polymerase Specific Activity Enzyme (units/μg) His₆ H-RT 281(1.0)^(a) His₆ H-H204R RT 325 (1.16) His₆ H-T306K RT 402 (1.43) His₆H-F309N RT 257 (0.91) His₆ H-M289L RT 339 (1.21) His₆ H-H204R T306K RT385 (1.37) His₆ H-H204R T306K F309N RT 391 (1.39) His₆ H-H204R M289LT306K F309N RT 410 (1.46) ^(a)Ratio of specific activity setting that ofHis₆ H-RT at 1.0.

His₆ H− F309N RT had a slightly reduced DNA polymerase specific activity(Table 10). The catalytic efficiency (k_(cat)/K_(m)) of His₆ H− RT andHis₆ H− H204R M289L T306K F309N were within a factor of two of eachother and their Kms for nucleotide substrate were similar (Table 11).

TABLE 11 Catalytic constants of purified RT mutants^(a) Enzyme K_(m)(μM) k_(cat) (sec⁻¹) k_(cat)/K_(m) His₆ H-RT 390 ± 98 45 ± 18 0.115 His₆H-H204R M289L 274 ± 92 46 ± 11 0.17 T306K F309N RT ^(a)Mean ± standarddeviation of three determinations

The monovalent and divalent metal ion optima of His₆ H− RT and His₆ H−H204R M289L T306K F309N RT were determined. CAT cRNA.DNA 24-mer inexcess over enzyme was used as template-primer (ExperimentalProcedures). The optima were the same: 75 mM KCl and 3 mM MgCl₂ at 37°C. for His₆ H− RT and His₆ H− H204R M289L T306K F309N and at 50° C. forthe quadruple point mutant. Taken together these results indicate thatthe four mutations identified that substantially increased the thermalstability of H-RT did not appreciably affect its DNA polymerasecatalytic capability.

In the presence of a template-primer, the half-life of M-MLV H− RT at50° C. is increased by a factor of 3 to 4 relative to the half-life inthe absence of nucleic acids. This is the result of the enzyme beingmore resistant to heat inactivation when bound to template-primer thanwhen unbound in solution. Mutations that impact the affinity of RT fortemplate-primer will affect the apparent thermal stability of RT when itis engaged in cDNA synthesis. Therefore the affinities (K_(D)) of His₆H− RT and several of its mutants for CAT cRNA.(dT)₂₀ were determined(Table 12).

TABLE 12 Nucleic acid dissociation constants of purified RT mutantsEnzyme K_(D) (nM)^(a,b) His₆ H-RT   8 ± 0.5 His₆ H-H204R RT  35 ± 0.5His₆ H-T306K RT 6.6 ± 0.4 His₆ H-F309N RT  28 ± 2.5 His₆ H-M289L RT 4.7± 0.7 His₆ H-H204R T306K RT 6.1 ± 1.5 His₆ H-H204R T306K F309N RT 7.3 ±2.4 His₆ H-H204R M289L T306K F309N RT  11 ± 1.0 ^(a)Mean ± standarddeviation of two or three determinations ^(b)The nucleic acid was CATcRNA•(dT)₂₀

The K_(D) of the His₆ H− RT quadruple mutant was increased somewhat from8 nM to 11 nM, indicating that nucleic acid binding affinity was reducedslightly by the four point mutations together. Interestingly two of thepoint mutations when present alone, H204R and F309N, reduced the bindingaffinity of His₆ H− RT substantially more, having a K_(D) of 35 and 28,respectively. The effects of these mutations on binding were apparentlycounter balanced by mutation T306K (K_(D)=6.6 nM), since the triplemutant H204R T306K F309N had a K_(D) of 7.3 nM. We conclude that whenthe triple (H204R T306K F309N) or quadruple mutant of His₆ H-RT isengaged in cDNA synthesis, any increase in apparent thermal stabilityobserved relative to His₆ H-RT is not due to increased protection byvirtue of binding more tightly to template-primer.

Practical Implication of Higher RT Thermal Stability—To assess thepractical impact of the increases in thermal stability imparted by theH− RT mutations selected in this study, we measured the ability of someof the mutants to synthesize full-length CAT cDNA between 40 and 55° C.(Table 13).

TABLE 13 Synthesis of full-length cDNA product from CAT cRNA by purifiedRT mutants at elevated temperatures^(a) Amount of Full Length Product(ng) Synthesized at Enzyme 40° C. 45° C. 50° C. 52.5° C. 55° C. His₆H-RT 517 523 229 40 2 His₆ H-H204R RT 521 446 146 36 17 His₆ H-T306K RT456 512 338 122 28 His₆ H-H204R T306K 664 528 330 202 43 RT His₆ H-H204RT306K 488 556 396 209 53 F309N RT His₆ H-H204R M289L 540 547 539 376 120T306K F309N RT ^(a)cDNA synthesis reaction mixtures (as described above)contained 9.3 pmoles (2.6 μg) of CAT cRNA. The amounts of full-lengthproducts were established by cutting the region corresponding to thesize of each full-length band from a dried alkaline 1.2% agarose gel andcounting it in a scintillation counter. Reaction mixtures contained 2pmoles of RT and were incubated at the temperatures indicated for 60min. The results of a single experiment are shown. Similar results wereobtained in one other experiment.

The amount of RT relative to template-primer was limiting in thesereactions. In the presence of limiting RT, failure to achievefull-length cDNA synthesis as the temperature is increased is anindicator of thermal inactivation of the enzyme under cDNA synthesisconditions. With one exception at temperatures above 50° C., the amountof full-length CAT cDNA synthesized by a particular RT mutant (Table 13)correlated well with the thermal stability (half-life) of the enzyme at50° C. (Table 9). The one exception is mutant H204R. It produced lessfun-length cDNA than His₆ H− RT at 50 and 52.5° C., in spite of having a5-fold greater half-life at 50° C. This is probably due to the weakerbinding of H204R than H− RT to CAT cRNA.DNA, diminishing thermalprotection by template-primer. All the RTs tested maintained activity at45° C. relative to 40° C., but with the exception of the quadruplemutant, all RTs synthesized less full-length CAT cDNA at 50° C. andabove. The His₆ H-RT quadruple mutant was fully active at 50° C. and at55° C. synthesized 60 times more full-length CAT cDNA than His₆ H− RT.This was 22% of the amount synthesized by both enzymes at 40° C.

The methods of the present invention can be used to engineer apolypeptide to have a, desired characteristic, for example, a desiredlevel of thermostability. The nucleic acid sequence of the polypeptidemay be sub-divided into segments and the segments individuallymutagenized, for example, using PCR random mutagenesis. The individualsegments may then be re-assembled and used to express a mutatedpolypeptide that is screened for the desired activity. The Examplesabove provide working embodiments of the invention in which the ratherlarge RT gene (2154 bases) was randomly mutated in segments by PCRrandom mutagenesis (Leung, et al., (1989) Technique 1, 11-15. Cadwell,et al., (1992) PCR Methods and Applications 2, 28-33). It may bedesirable to sub-divide the sequence of the polypeptide of interest intofragments that correspond to functional or structural domains of thepolypeptide of interest if such domains are known. In the workingembodiments above, the segments roughly corresponded to the codingsequences of the five separate structural subdomains of RT (Kohlstaedt,et al., (1992) Science 256, 1783-1789. Jacobo-Molina, et al., (1993)Proc. Natl. Acad. Sci. USA 90, 6320-6324). Segments one through fourcorresponded to the polymerase subdomain of fingers, palm, thumb, andconnection, respectively, and segment five corresponded to the RNase Hdomain (FIG. 2A).

Using the materials and methods of the present invention, one skilled inthe art can construct a modified RT enzyme having a desired level ofthermostability. In the examples presented above, the operatingtemperature of H− M-MLV RT was increased to at least 10° C. above 45° C.This target temperature was selected as a compromise: high enough tohelp reduce RNA secondary structure and low enough to avoid RNAbreakdown. Although, the rate of chemical RNA breakdown catalyzed byMg⁺⁺ increases dramatically as the temperature is increased above 55 to60° C. under cDNA synthesis reaction conditions, it may be desirable incertain instances (e.g., RNA with pronounced secondary structure) toconduct a reverse transcription reaction at a temperature higher than55° C., for example, at about 60° C., or at about 65° C., or at about70° C. Using the methods described above a suitable RT can beconstructed using routine experimentation.

Reaction conditions for the mutagenesis reaction may be adjusted tointroduce the desired number of mutations in each segment. In thepresent study, an upper limit cut-off of 1 to 2 mutations per segmentwas set as the target for mutation frequency to suppress accumulation ofdeleterious mutations (Lehmann, et al., (2001) Current Opinion inBiotechnology 12, 371-375), and to minimize the amount of screeningrequired to find active mutants. Those skilled in the art willappreciate that conditions may be adjusted in order to induce a greatermutation frequency, for example, by adjusting the concentration of Mn²⁺,pH, salt concentration etc. Mutation frequencies of >5 mutations/segmentin segment one, two, or three produced only about 5% active mutants withall the mutants having less thin wild-type activity. At the mutationfrequency used of 1 to 2 mutations per segment, approximately one-thirdof the mutants had little or no activity, one-third had less than 50% ofHis₆ H-RT activity, and one-third had up to 100% of His₆ H-RT activity.

Polypeptides produced from the mutated sequences are screened for adesired activity. The polypeptides may be screened from crude extracts,partially purified extracts or from purified polypeptide preparations.In the Examples above, crude extracts of mutants were screened in a96-well plate format with template-primer specific for RT activity(Gerard, et al., (1974) Biochem. 13, 1632-1641). The procedure used toproduce E. coli cell crude extracts permeabilized rather the lysed thecells, releasing proteins and small RNAs to the outside of the cellmatrix, while keeping DNA inside. This made representative sampling andassay with an exogenous template-primer of the cell suspension feasible.The signal-to-noise ratio of the assay was such that to differentiatestarting from mutant cells at least a two-fold difference in activitywas required. Those skilled in the art will appreciate that othermethods of screening, for example, by first purifying the mutatedpolypeptides of interest (e.g., RTs) will permit detection of smallerchanges in activity level.

Utilizing this approach, two mutations were found in segment three(thumb) and one in segment two (fingers). A rational sequence homologyapproach combined with site-directed mutagenesis was used to identify afourth mutation located in the fingers subdomain. The small number ofmutations found and the failure to identify mutations in segments oneand four were probably due to limitations of both the random mutagenesisapproach and the screen. The mutation frequency cut off of 1 to 2mutations per segment imposed on the random mutagenesis limited themutant space available in the mutant population. The inability of theassay to detect less than a two-fold difference in activity probablymade many active and slightly more thermal stable mutants undetectable.Since the RNase H domain of wild-type M-MLV RT is already substantiallymore thermal stable than the polymerase domain (Verma, I. (1975) J.Virol. 15, 843-854), it was unlikely that stabilizing mutationsintroduced in segment five would be picked up in a screen for morethermal stable polymerase activity.

In spite of the limited number of mutations found with the approachused, those that were identified had a significant positive impact on RTthermal stability. The thermal stability of His₆ H− M-MLV H204R M289LT306K F309N RT was increased 30 fold at 50° C. The RT operatingtemperature was increased 7° C. to 55° C. Three of the four mutationsalone increased the RT half-life 1.2 to 1.6-fold and the fourth mutationgave a 5-fold increase. A well established operating principle inprotein stabilization is that the total stabilization obtained bymultiple mutations is the sum of the effects of individual mutations,assuming each mutation exerts its effect independent of the othermutations (Wells, J. A. (1990) Biochem. 29, 8509-8517). The thermalstabilizing effects of the four mutations appear to be additive, but anadditional contribution to increased thermal stability due tocooperative interaction between mutations cannot be excluded.

Discussion of the mechanisms whereby each of these four mutations affectthe thermal stability and template-primer binding affinity of M-MLV RTrequires a 3-dimensional structural framework. In the case of themutation at H204 in the M-MLV RT palm subdomain, this is easilyaccomplished because the structure of a fingers-palm fragment of M-MLVRT is known (Georgiadis, et al., (1995) Structure 3, 879-892). For T306Kand F309N located in the thumb, we must rely on 3-dimensional structuraldata taken from HIV RT (Kohlstaedt, et al., Jacobo-Molina, et al., Ding,et al., (1998) J. Mol. Biol. 284, 1095-1111, and Huang, et al., (1998)Science 282, 1669-1675). Fortunately in this region of the thumbsubdomain of HIV RT (T253 to P272) and the corresponding region of M-MLVRT (T296 to P315), the amino acid sequence is reasonably well conserved.M-MLV RT can be predicted to have an α-helix in this region thatresembles the structure of HIV RT α-helix H (Kohlstaedt, et al.,Jacobo-Molina, et al., Kneller, et al., (1990) J. Mol. Biol. 214,171-182, and Rost, B. (1996) Methods Enzymol. 266, 525-539.)

Together the four RT amino acid changes exerted their stabilizing effectwithout significantly altering the DNA synthesizing catalytic activityof the enzyme. However two mutations, H204R and F309N, did significantlyreduce RT binding to template-primer (Table 12). M-MLV RT H204R islocated in the middle of the long α-helix H (aH) on the backside of thepalm subdomain, directly below β-sheets 10-11 (β10-β11) (FIG. 9,Georgiadis, et al., supra). Beta-sheets 10-11 contain the M-MLV RTpolymerase catalytic site (YVDD) at residues 222 to 225 on the inside ofthe palm that contact the template-primer (Georgiadis, et al., supra).Changes in H204R could directly affect packing of amino acid residuesnear it in αH and β10-β11, thus influencing how β10-β11 interact withtemplate-primer. Or its effect could be translated along the polypeptidebackbone since the α-helix containing H204 and the β-sheet containingY222 are separated by a single short turn (FIG. 9). As discussed abovebased upon an amino acid homology comparison of M-MLV and HIV RT andfrom crystal structures of HIV RT (Kohlstaedt, et al., supra,Jacobo-Molina, et al., supra, Ding, et al., supra, Huang, et al., supra,Kneller, et al., supra, Rost, Bebenek, et al., (1997) Nature StructuralBiology 4, 194-197, and Beard, et al., (1998) J. Biol. Chem. 273,30435-30442), F309 in M-MLV RT can be predicted to be part of a minorgroove binding track in an α-helix of the RT thumb subdomain. In HIV RTthis structure contributes significantly to RT binding totemplate-primer (Bebenek, et al., supra). The aromatic phenylalanine atposition 309 in M-MLV RT corresponds to tryptophan 266 in HIV RT(Bebenek, et al., supra). Extensive amino acid substitution studies ofHIV RT W266 show that any substitution at this position reduces thebinding affinity of RT for template-primer (Beard, et al., supra). Wepostulate that alteration of M-MLV RT F309 to asparagine impacts thestructure of a putative M-MLV RT minor groove binding track resulting ina reduction in binding affinity for template-primer. Interestingly T306Kwas able to compensate for the reduced binding produced by H204R orH204R and F309N together, restoring template-primer binding affinity tothe original level (Table 12). Again based upon amino acid homology inthis region, T306 in M-MLV RT corresponds to a lysine in position 263 inthe α-helix of HIV RT that contains the minor groove binding track. Thecrystal structure of HIV RT complexed with double-stranded DNA showsthat K263 contacts the n-2 phosphate of the DNA primer (Ding, et al.,supra, Huang, et al., supra). Introduction into M-MLV RT of a positivelycharged lysine in place of phenylalanine at position 306 compensates forthe reduced binding of H204R and F309N, perhaps by enhancing RT bindingat the n-2 primer phosphate position.

H204R had the greatest individual thermal stabilizing effect on H− M-MLVRT. At position 204 histidine has been replaced with a more highlycharged arginine in α-helix H at the underside of the palm subdomain(Georgiadis, et al., supra). Examination of the 3-dimensional structuralmodel of the fingers-palm fragment of M-MLV RT (Georgiadis, et al.,supra) reveals this substitution places arginine at a proper distance toform a salt bridge with E201 in α-helix H or a hydrogen bond with T128in the loop between β-sheet 6 and α-helix E (FIG. 9). Either event wouldhelp stabilize the RT molecule (Kumar, et al., (2001) Cell. Mol. Life.Sci. 58, 1216-1233).

The mutation at position 306 from threonine to lysine probablycontributes to the thermal stability of M-MLV RT by making anelectrostatic contribution to stabilizing the polypeptide backbone.Saturation of this site with all possible amino acids showed that onlysubstitution of arginine or lysine increased M-MLV RT thermal stability.Both of these amino acids could allow for hydrogen bonding to main chaincarbonyl groups and amino acid side chains in a loop adjacent to theputative a-helix containing residue 306.

Predictions about the mechanism whereby F309N increases M-MLV RT thermalstability are complicated by its proposed role in template-primerbinding (see discussion above). M-MLV RT position 309 was also examinedby saturation with all possible amino acid changes. Only serine andasparagine increased thermal stability. Substitution of charged aminoacids for the exposed hydrophobic phenylalanine, which in other proteinfamilies correlates well with amino acid changes observed intransitioning from mesophilic to thermophilic family representatives(Kumar, et al., supra, Cambillau, et al., (2000) J. Biol. Chem. 275,32383-32386), reduced or eliminated polymerase activity. Correlation ofincreased thermal stability with substitution of polar for exposedhydrophobic amino acids is observed much less frequently in proteinfamilies (Kumar, et al., supra, Cambillau, et al., supra), so that thestructural basis for the stabilizing effect of F→N at position 309 isnot clear.

In the absence of both 3-dimensional structural information andreasonable sequence homology between HIV RT and M-MLV RT in the regionof M-MLV RT M289, it is difficult to predict the structural basis forthe contribution of M289L to M-MLV RT thermal stability. As a generaltendency, thermally stable proteins have more and larger hydrophobicamino acids than their mesophilic counterparts that are better able toexclude water from the protein core (Cambillau, et al., supra). It ispossible that M→L at position 289 increases M-MLV RT thermal stabilitythrough allowing better hydrophobic packing by replacing an unbranchedmethionine with a β-branched leucine (Cambillau, et al., supra).

Consistent with the results of other studies focused on identifyingmutations that increase protein thermal stability (Arnold, et al.,(2001) Trends in Biochemical Sciences 26, 100-106), the mutationsidentified in this study probably reside on the RT surface and influencethermal stability without decreasing catalytic activity. In the case oftwo of the stabilizing amino acids, T306 and F309, they probably resideon a protein surface that contacts template-primer and they bind in sucha way that their effects on binding are off setting.

The present invention provides materials and methods for engineering apolypeptide to contain a desired characteristic. As an example, theM-MLV RT gene was mutated to increase the thermostability of the enzyme.The goal of this research was to increase the temperature at which M-MLVRT efficiently catalyzes cDNA synthesis from 48° C. to about 55° C. toabout 60° C. by increasing the intrinsic thermal stability of RT. Theability to use retroviral RT at 55 to 60° C. increases both theefficiency and specificity of priming of cDNA synthesis. With themutagenesis and screening approaches used, mutations were identifiedthat increased the M-MLV RT operating temperature limit to at leastabout 55° C. These mutations created a dramatic increase in intrinsicthermal stability measured as a half-life increase at 50° C. (30-fold),that was maintained partially at 55° C. (5-fold increase in hallife).There are alternative approaches that could potentially build on thefour mutants already identified. For example, the target mutationfrequency could be set much higher (5-10 mutations/segment) duringrandom mutagenesis of several larger segments of the H-M-MLV RTquadruple mutant polymerase domain, giving a much smaller proportion ofactive mutants but a much broader mutant spectrum. Several librariesgenerated in this fashion could then be shuffled by recombination(Stemmer, W. P. C. (1994) Nature 370, 389-391), which would tend toeliminate deleterious mutations (Lehmann, et al., supra). When combinedwith a 96-well DNA polymerase activity screen made more sensitive and asampling procedure used to assay multiple mutants per well to increasethe number of mutations screened, such an approach should yieldadditional mutations that further increase the thermal stability ofM-MLV RT.

Example 7 Engineering of SUPERSCRIPT™ III RT, Thermal Stability, cDNASynthesis

The gene for Moloney murine leukemia virus (M-MLV) RNase H-minus (H-)reverse trenscriptase (RT), also known as SUPERSCRIPT™ II was randomlymutagenized and mutants were screened for increased thermal stability.Four mutations were identified that together increased the half-life ofM-MLV H− RT at 50° C. 35-fold and raised the RT cDNA synthesis operatingtemperature from 42° C. to 55° C. This increase in thermal stability wasachieved by increasing the intrinsic thermal stability of M-MLV H− RTand without diminishing the DNA polymerase catalytic activity of theenzyme.

Reverse transcriptase (RT, e.g., retroviral RT) is an essential tool forthe synthesis and cloning of cDNA. Forms of retroviral RT used widely tosynthesize cDNA are M-MLV RT and AMV RT. In addition to polymeraseactivity, retroviral RT possesses RNase H activity that degrades the RNAin an RNA-DNA hybrid (Moelling, et al., (1971) Nature New Biology 234,240-244). The presence of this degradative activity is responsible inpart for the limitation on efficient synthesis of long cDNA (Krug andBerger (1987) Methods in Enzymol. 152, 316-325; Berger, et al., (1983)Biochem. 22, 2365-2372). The RNase H domain of RT can be mutated toreduce or eliminate RNase H activity while maintaining mRNA-directed DNApolymerase activity (Kotewicz, et al., (1988) Nuc. Acids Res. 16,265-277; DeStefano, et al., (1994) Biochim. Biophys. Acta 1219,380-388), improving the efficiency of cDNA synthesis (Kotewicz, et al.,supra). This has been done in SUPERSCRIPT™ II and ThermoScript.

A second significant drawback to copying mRNA is the tendency of RT topause during cDNA synthesis resulting in the generation of truncatedproducts (Harrison, et al., (1998) Nuc. Acids Res. 26, 3433-3442;DeStefano, et al., (1991) J. Biol. Chem. 266, 7423-7431). This pausingis due in part to the secondary structure of RNA (Harrison, et al.,supra, Wu, et al., (1996) J. Virol. 70, 7132-7142). Performing cDNAsynthesis at reaction temperatures that melt the secondary structure ofmRNA helps to alleviate this problem (Myers and Gelfand, (1991) Biochem.30, 7661-7666). In addition, the oligo(dT)n primer often used toinitiate cDNA synthesis tends to prime at internal stretches of Aresidues in mRNA at lower temperatures, resulting in the synthesis of3′-end truncated cDNA products. M-MLV RT does not efficiently synthesizecDNA from mRNA above 43° C. (Tosh, et al., (1997) Acta Virol. 41,153-155).

In an effort to raise the temperature at which SUPERSCRIPT™ II RT can beused to synthesize cDNA, we have randomly mutagenized the SUPERSCRIPT™II RT gene and screened for thermal stable mutants. Several thermalstable mutants of SUPERSCRIPT™ II RT were identified and purifiedenzymes were characterized. We show that when the mutations are presenttogether they increase RT thermal activity by increasing its intrinsicthermal stability without altering catalytic activity.

SUPERSCRIPT™ III RT Purification: The SUPERSCRIPT™ III RT gene wasderived from SUPERSCRIPT™ II RT. Four mutations, in addition to the 3RNase H mutations present in SUPERSCRIPT™ II, were included along withmodifications to the N-terminus to increase the thermostability. Thegene was cloned into plasmid pBAD (Invitrogen Corporation, Carlsbad,Calif.) under control of an araD promoter. The purified protein has anapparent molecular weight of 78 kDa. The plasmid was transformed into E.coli DH10B (Invitrogen Corporation, Carlsbad, Calif.) cells and a seedstock was grown up to an O.D. of 1.0 in EG (2% Tryptone, 1% yeastextract, 0.5% glycerol, 10 mM NaCl, and 1 mM KCl) and frozen in 60%S.O.B./40% glycerol at −80° C.

For purification, a stab from the frozen seed was used to inoculate astarter culture of 500 ml. The media used for the R&D prep was either EGor CircleGrow (Bio101), supplemented with 100 μg/ml Ampicillin. Thestarter culture was incubated overnight at 37° C. and used to inoculate10 liters EG+Amp media. The culture was incubated at 37° C. to an OD₆₀₀of 1.0 (3-4 hours), induced with 0.2% arabinose, and incubationcontinued another 3 hours. Cells were pelleted by centrifugation at5,000 g for 20 minutes at 4° C. The supernatant was removed and cellswere either frozen at −80° C. or processed.

Purification of SUPERSCRIPTT™ III was done the same as SUPERSCRIPT™ IIand is outlined as follows, with the exception of using an AF-Heparin650M resin (TosoHaas) in place of Heparin Agarose as the third column.All buffers must be autoclaved and columns thoroughly cleaned to preventRNase contamination. The procedure is done at 4° C. unless otherwiseindicated.

Purification Method A. Thaw 50 grams of cells in 100 ml Buffer A (2 mlper g of cell pellet: 20 mM Tris-HCL pH 7.5, 25 mM NaCl, 1 mM EDTA, 1 mMDTT, 1.3 mM PMSF), stirring at 4° C. until homogenous. Add another 0.78ml 0.1 M PMSF and mix well (5 minutes). Strain cells through 6 layers ofcheesecloth. Filter the sample through gaulin pre-equilibrated with 1volume Buffer A (avoid bubbles and particulates in gaulin) at 4-6° C.with pressure at 8000 psi. Repeat and check sample for >80% lysis.Determine volume of cells and add 0.053 V of 5M NaCl to lysate. Stir at4° C. till mixed. Add 0.111 V of 5% polymin P (pH 7.9) over 30 minuteswhile mixing and then mix for another 30 minutes. Centrifuge at 18,100 gfor 60 min at 4° C., keep the supernatant. Add 226 g/L (NH₄)₂SO₄ tosupernatant over 30 minutes while stirring and mix for another 30minutes. Centrifuge at 18,100 g for 60 min at 4° C., keep thesupernatant. Resuspend the pellet in Buffer B (20 mM Tris-HCL pH 7.5,100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NOG, 5% glycerol) at 0.52 ml/goriginal pellet weight (26 ml) while stirring for 1 hour.

A G-25 F column (AP-Biotech, 270 ml resin volume) was equilibrated withBuffer B and then the sample was loaded. The column was washed and thesample eluted with 2 column volumes of Buffer B at a flow rate of 3.3ml/min and fraction volume of 6 ml/tube. Fractions were pooled based onUV absorbance and a clear amber color. If precipitation formed, the poolwas centrifuged at 18,100 g for 30 min at 4° C.

The fraction pool was loaded onto a P-11 column (Whatman: 2× cycled, 25ml resin volume) pre-equilibrated with Buffer B at a flow rate of 1.0ml/min. The column was washed with 10 column volumes of Buffer C (20 mMTris-HCL pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 5%glycerol) and eluted with 12 column volumes of a 100 to 500 mM NaClgradient (Buffer C and 0 to 100% of Buffer D: 20 mM Tris-HCL pH 7.5, 500mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 5% glycerol) at a flow rateof 1.6 mL/min and fraction volume of 4 ml. Fractions were pooled byeither OD₂₈₀>50% peak height or >50% peak RT activity. The fraction poolwas then diluted with one volume of Buffer E (20 mM Tris-HCL pH 7.5, 1mM EDTA, 1 mM DTT, 0.01% NP-40, 5% glycerol).

The diluted pool was then loaded onto an AF-Heparin 650M column(TosoHaas, 12.5 ml resin volume) pre-equilibrated with Buffer F (20 mMTris-HCL pH 7.5, 160 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 5%glycerol) at a flow rate of 0.7 mL/min.

The column was washed with 10 column volumes of Buffer F (20 mM Tris-HCLpH 7.5, 160 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 5% glycerol) andeluted with 10 column volumes of a 160 to 700 mM NaCl gradient (Buffer Fand 0 to 100% of Buffer G: 20 mM Tris-HCL pH 7.5, 700 mM NaCl, 1 mMEDTA, 1 mM DTT, 0.01% NP-40, 5% glycerol) at a flow rate of 0.5 mL/minand fraction volume of 2 ml. Fractions were pooled by either OD₂₈₀>50%peak height or >50% peak RT activity. The fraction pool was then dilutedwith three volumes of Buffer H (20 mM Tris-HCL pH 7.5, 100 mM NaCl 1 mMEDTA, 1 mM DTT, 0.03% NP-40, 5% glycerol).

The diluted pool was then loaded onto an SP Sepharose HP column (ApBiotech, 12.5 ml resin volume) pre-equilibrated with Buffer I (20 mMTris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% NP-40, 5%glycerol) at a flow rate of 1.2 ml/min. The column was washed with 10column volumes of Buffer I (20 mM Tris-HCL pH 7.5, 100 mM NaCl, 1 mMEDTA, 1 mM DTT, 0.01% NP-40, 5% glycerol) and eluted with 10 columnvolumes of a 100 to 280 mM NaCl gradient (Buffer I and 0 to 40% ofBuffer G) at a flow rate of 0.6 mL/min and fraction volume of 2 ml.Fractions were pooled by either OD₂₈₀>50% peak height or >50% peak RTactivity.

The RT containing fractions from the Sepharose column were dialyzedagainst 20 volumes Buffer J (20 mM Tris-HCL pH 7.5, 100 mM NaCl, 0.1 mMEDTA, 1 mM DTT, 0.01% NP-40, 50% glycerol) overnight at 4° C.

Purification Method B. One hundred grams of cells is suspended in 80 mlof permeabilization buffer (500 mM Bis-Tris, pH 7.0 at 4° C./50 mMEDTA/5 mM DTT). (Note: The DTT can be added as a separate component andthe volume of water added adjusted accordingly.) 79.6 ml of H₂O is addedand the cell suspension is stirred with an overhead stirrer at ˜700 rpmwith a Heidolph mixer or at 6+ with a StirPak mixer using the threeblade mixer shaft. The suspension is checked to ensure that it ishomogeneous by stopping the mixer and pouring the suspension into aglass beaker slowly while looking for clumps. Once thoroughly mixed, 80ml 25% Triton X-100/10% deoxycholic acid (at ambient temperature) isadded while mixing vigorously (as above). 400 ul of 100 mM PMSF is addedand the suspension is allowed to stand with mixing for 30 min at 4° C.60 ml of 2 M (NH₄)₂SO₄ is added and mixing is continued for anadditional 15 min at 4° C. Samples can now be taken or proceed to thefiltration stage.

Volumes: Cells 100 ml Buffer 80 ml H₂O 79.6 ml Detergent 80 ml PMSF0.400 ml (NH₄)₂SO₄ 60 ml 400 ml

Filtration. The cell slurry is poured into a circulation vessel and aflow rate of 1.5 L/min/ft2 through a 0.2 um Spectrum mixed estercellulose hollow fiber filter with a 1.0 mm lumen is established.Extraction is performed with seven volumes of 100 mM TRIS, pH 8.0/300 mMammonium sulfate/10 mM EDTA/1 mM DTT/10% glycerol (v/v) whilemaintaining 4-6° C. and a TMP of 5-10 psi.

Once enough filtrate has been collected, the second stage filtrationusing an AG/Tech 30,000 MWCO hollow fiber with a maximum inlet pressureof 50 psi. (all operations chilled to 4-6° C.) is initiated. Once allseven volumes of extraction have been collected, the solution isconcentrate to 600 ml and diafiltered against five volumes of 100 mMTris, pH 8.0/100 mM NaCl/10 mM EDTA/1 mM DTT.

The diafiltered retentate is collected and 50 ml previously equilibratedDEAE cellulose is added. The suspension is mixed gently for 15 min at 4°C. The resulting solution is clarified by filtration through a CUNO 30SP depth filter.

Chromatography. The above solution is applied to a previouslyequilibrated 120 ml column of Macroprep High S (20 mM Tris, pH 8.0/150mM NaCl/10% glycerol/1 mM EDTA/1 mM DTT/0.01% Triton X-100). The columnis washed with 10 column volumes of equilibration buffer and eluted witha linear gradient of equilibration buffer to the same buffer at 800 mMNaCl. 50 fractions are collected and the major uv peak is pooled.

The pool is applied to a previously equilibrated 120 ml column ofCeramic hydroxy apatite (20 mM Kpi, pH 7.0/100 mM KCl/10% glycerol/1 mMDTT/0.01% Triton X-100) at 20 cm/h. Elution is with this same buffer and10 ml fractions are collected. The uv peak is pooled and an equal volumeof solubilization buffer (100 mM Tris, pH7.5/300 mM NaCl/0.2 mM EDTA/30%glycerol/1 mM DTT) is added.

The pool is applied to a previously equilibrated 16 ml column of EM COO—(20 mM Tris, pH 7.5/100 mM NaCl/0.1 mM EDTA/20% glycerol/1 mM DTT/0.01%Triton X-100) at 20 cm/h. The column is washed with two column voleculesof equilibration buffer. Elution is with a linear gradient ofequilibration buffer to the same buffer with 200 mM NaCl. The uv peak ispooled and dialyzed against 20 volumes of 20 mM Tris, pH 7.5/100 mMNaCl/0.1 mM/1 mM DTT/0.01% Triton X-100/50% glycerol.

DNA Polymerase Assays: RT DNA polymerase unit activity was assayed with(rA)₆₃₀.p(dT)₁₂₋₁₈. One unit of DNA polymerase activity is the amount ofRT that incorporates one nmole of deoxynucleoside triphosphate into acidinsoluble product at 37° C. in 10 min. Specific Activity is determinedby dividing the Units/μl by the protein concentration to get units/mg.

cDNA synthesis from CAT cRNA was carried out in reaction mixtures (20μl) containing 50 mM Tris-HCl (pH 8.4), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiotreitol (DTT), 500 μM each of dATP, dTTP, dGTP, and [α-³²P]dCTP(300 cpm/pmole), 1,750 units/ml RNase Inhibitor, 130 μg/ml (465 nM) CATcRNA, 20 μg/ml (2,300 nM) p(dT)₂₅₋₃₀, and 3,250 units/ml (100 nM) RT.Incubation was at various temperatures for 60 min individual tubes. Analiquot of the reaction mixture was precipitated with TCA to determineyield of cDNA synthesized, and the remaining cDNA product was sizefractionated on an alkaline 1.2% agarose gel. To establish mono- anddivalent metal reaction optima, initial reaction rates were determinedunder conditions of limiting RT concentration during 10 min incubationat 37° C. or 50° C. Reaction mixtures (20 μl) contained 50 mM Tris-HCl(pH 8.4), 10 mM DTT, 500 μM each of dTTP, dATP, dCTP, and [³H]dGTP (100cpm/pmole), 10 pmoles (2.8 μg) CAT cRNA, 50 pmoles DNA 24-mer, 0.5pmoles RT, and KCl and MgCl₂, varied in concentration one at a time.

Half Life Determination:

Mixtures (20 μl) were incubated for various times in 0.5-ml tubes in athermocycler at 50° C. or 55° C. and contained 50 mM Tris-HCl (pH 8.4),75 mM KCl, 3 mM MgCl₂, 10 mM DTT, OA % (v/v) Triton X-100, and 3-7 μg/mlpurified RT. Incubation was stopped by placing the tube in ice. Analiquot (5 μl) was assayed for residual activity with(rA)₆₃₀.p(dT)₁₂₋₁₈.

Measurement of K_(D) by Filter Binding:

A nitrocellulose filter-binding assay was used to determine the nucleicacid binding constants (K_(D)) of RTs for CAT cRNA.(dT)₂₀.Dephosphorylated CAT cRNA was labeled at the 5′ end with [γ-³²P]ATP andT4 polynucleotide kinase (Boehringer). Oligo(dT)₂₀ was annealed to thepoly(A)-tailed CAT cRNA in a buffer containing 10 mM Tris-HCl, pH 7.5,and 80 mM KCl at 65° C. for 5 min followed by chilling on ice. Reactionmixtures (100 μl) containing binding buffer (50 mM Tris-HCl, pH 8.4, 75mM KCl, 3 mM MgCl₂, and 10 mM DTT), 0.05 nM ³²P-labeled CAT cRNA, 1 nM(dT)₂₀, and 1 to 50 nM RT were incubated at 23° C. for 5 min. Afterincubation, the mixture was filtered through a nitrocellulose filter(Millipore, HA 0.45 mm) soaked in binding buffer, which was then washedwith binding buffer. The K_(D) is equal to that enzyme concentration atwhich 50% of the labeled CAT cRNA is bound. For this method of analysisto be valid, the CAT cRNA concentration in the reaction must besubstantially below K_(D), so that the total enzyme concentrationapproximates the concentration of free unbound enzyme.

LacZα Fidelity Measurements:

Fidelity measurement were conducted using a modified lacZα gappedprocedure from Boyer, J. C. et al., (Methods in Enzymology, Vol 275, p523-537). Briefly, a modified pUC19 plasmid containing the promoter forT7 RNA polymerase between nucleotides −112 and −113 (where position +1is the first transcribed nucleotide of the lacZα gene) was used. Theplasmid was linearized with FspI, and transcription by T7 RNA polymeraseproduces a 344 nucleotide transcript. Using this RNA as template, cDNAsynthesis under desired conditions (200 units SUPERSCRIPT™ III RT or 15units AMV H+ RT in their respective buffers) was initiated from a 15-merDNA primer. After heat denaturation, the RNA was digested and an excessof cDNA was used for hybridization to a circular DNA substratecontaining a complementary single-stranded gap. This substrate was madeby cutting M13mp19 RF with PvuI and PvuII in the appropriate buffer andisolating the large linear fragment. This fragment is then denatured andreannealed to a 2-fold excess of single-strand M13mp19. The resultingDNA product is transfected into competent DH12S cells, plated onto LBindicator plates containing X-gal and IPTG and plaques are scored forcolor. Mutation frequency was determined by dividing the number ofmutant plaques (light blue or white) by the total. The enzyme's mutationfrequency is corrected by subtracting the mutation frequency of thestarting DNA. The error rate is determined by dividing the mutationfrequency by the number of known detectable sites (116) and thendividing by the probability of expressing the minus strand (50%).

rpsL Fidelity Assay:

Fidelity assay was performed based on the streptomycin resistance thatrpsL mutatants exhibit (Fujii, et al., (1999) J Mol Biol.289(4):835-50). Briefly, pMOL 21 plasmid DNA (4 kb), containing theampicillin (Ap^(r)) and (rpsL) genes, was modified with the insertion ofa T7 RNA promoter sequence downstream of the rpsL gene. The plasmid waslinearized with ScaI and this template was used for in vitrotranscription of a 1.4 kb RNA containing the rpsL gene. A first strandcDNA reaction using the desired RT and reaction conditions was carriedout, followed by second strand synthesis using pol I, RNase H andligase. This double-strand DNA was then cut with HindIII and EcoRI andcloned back into the original pMOL 21 plasmid and transformed into MF101competent cells. Cells were plated on ampicillin plates to determine thetotal number of transformed cells. Cells were plated on ampicillin andstreptomycin plates to determine the total number of rpsL mutants.Mutation frequency was determined by dividing the total number ofmutations by the total number of transformed cells. The error rate wasdetermined by dividing the mutation frequency by 130 (the number ofamino acids that cause phenotypic changes for rpsL) and the templatedoubling. In addition rpsL was further modified by altering the sequenceprone to mutations to remove template bias.

TDT Activity:

The template-primer was prepared by annealing a 47-mer template (eitherRNA or DNA):

5′-GAGTTACAGTGTTTTTGTTCCAGTCTGTAGCAGTGTGTGAATGG

AAG-3′ (SEQ ID NO:6) to a 18-mer DNA primer (5′-GAACAAAAACACTGTAACTC-3′(SEQ ID NO:10)) [³²P]-labeled at the 5′-end with T4 polynucleotidekinase (template:primer, 3:1). Assay mixture (10 μl) contained 5 nMtemplate-primer, 200 units each of either SUPERSCRIPTT™ II orSUPERSCRIPT™ III, all 4 dNTPs, none or dCTP, dATP, dTTP, dGTP (250 uMeach) as shown in the figure legends, 50 mM Tris-HCl (pH 8.3), 75 mMKCl, 3 mM MgCl₂, 10 mM DTT. Reactions were incubated at varioustemperatures for 10 or 60 min and terminated by the addition of 5 μl of40 mM EDTA, 99% formamide. Reaction products were denatured byincubating at 95° C. for 5 min and analyzed by electrophoresis on urea6% polyacrylamide gels.

Half Life Determination:

Mixtures (20 μl) were incubated for various times in 0.5-ml tubes in athermocycler at 50° C., 55° C., or 60° C. and contained 1× First StrandBuffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂), 10 mM DTT, 3 mMMgCl₂ (total to 6 mM), 0.5 mM dTTP, 0.05% (v/v) NP-40, [methyl-³H]dTTP(20 μCi/ml), and 5 μl of RT. Incubation was stopped by placing the tubein ice. An aliquot (5 μl) was assayed for residual Unit activity with 5mM poly(A)/3 mM oligo(dT)₂₅. One unit of RT DNA polymerase activity isthe amount of RT that incorporates 1 nmol of deoxynucleosidetriphosphate into acid-insoluble product at 37° C. in 10 min.

Full-Length cDNA Profiling:

cDNA synthesis from cRNA by SUPERSCRIPT™ III was carried out in reactionmixtures (20 μl) containing 1× First Strand Buffer (50 mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM MgCl₂), 10 mM dithiotreitol (DTT), 500 μM each ofdATP, dTTP, dGTP, and dCTP, [α-³²P]-dCTP (300 cpm/pmole), 2 units/μlRNaseOUT, different amounts of RNA, and 0.5 μg oligo(dT)₂₅. The mix isprewarmed at various temperatures for 2 minutes. After warming, 200 or400 units of SUPERSCRIPT™ III RT are added. The mixtures were incubatedat the same temperature for another 50 min in individual tubes. Thereactions were stopped by adding 10 μl of 0.5 M EDTA. An aliquot (5 μl)of the reaction mixture was precipitated with TCA to determine yield ofcDNA synthesized, and the remaining cDNA product was size fractionatedon an alkaline 1.2% agarose gel (McDonell, et al., J. Mol. Biol. 110,199-146, 1977). For the RTs from other vendors, each was assayedfollowing the manufacturer's suggestion.

Results and Discussion

Designation of polypeptides. Unless otherwise indicated, the followingdesignations will be used to describe various polypeptides of theinvention. SUPERSCRIPT™ II is M-MLV reverse transcriptase with amethionine added as a start codon (as discussed earlier, wild type M-MLVRT is a cleavage product that does not contain a methionine) and havingthree point mutations that reduce RNase H activity. EFN indicates apolypeptide having the SUPERSCRIPT™ II sequence with an N-terminal sixhistidine tag sequence MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKH (amino acids1-32 of SEQ ID NO:2 and Table 3) and containing the EFN mutations, whichare H204R, T306K, and F309N of the SUPERSCRIPT™ II sequence. Tagged, noHis EFN has the tag sequence MASGTGGQQMGRDLYDDDDKH (SEQ ID NO:3) and theEFN mutations, no tag EFN contains the EFN mutations. SUPERSCRIPT™ III,which may be designated LEFN, has the SUPERSCRIPT™ II sequence and anN-terminal tag sequence MASGTGGQQMGRDLYDDDDK (SEQ ID NO:11) and the LEFNmutations, which are H204R, M289L, T306K, and F309N of the SUPERSCRIPT™II sequence. His tagged LEFN indicates a polypeptide having anN-terminal six histidine tag sequence MGGSHHHBHHGMASMTGGQQMGRDLYDDDDKH(amino acids 1-32 of SEQ ID NO:2 and Table 3) and containing the LEFNmutations.

Purification:

The protein expresses well and appears to not be toxic. Varying levelsof arabinose induction were used with 0.2% being optimal but not verydifferent from 2.0%. Induction times longer than 3 hours can be done,however there is a 70 kDa product that is generated over longerincubation periods. It appears to be a proteolysis product removing theC-terminus as purified N-terminal Histidine-tagged clones also had thisprotein co-purify.

The purification protocol is essentially the same as was used forSUPERSCRIPT™ II and the SUPERSCRIPT™ III enzyme behaves almost the samethroughout the purification. SUPERSCRIPT™ III may be prepared atconcentrations of 2,000-4,000 units/μl after dialysis with noprecipitation problems.

Fidelity:

Initial results have shown no difference between SUPERSCRIPT™ II andSUPERSCRIPT™ III using either the rpsL or lacZα fidelity assay. RTs havesequence dependant mutation hotspots in homopolymeric runs ofnucleotides, primarily runs of A or T. These most common mutations atthese runs are one nucleotide insertion or deletion which is caused bythe template or primer breathing and re-annealing with an overlap. Theresulting frameshift mutation usually results in premature terminationof the gene product. The modified EFN polypeptide, which contains theH204R, T306K, and F309N mutations, had improved frameshift error ratescompared to SUPERSCRIPT™ II and MMLV H+. Experiments are ongoing todetermine if SUPERSCRIPT™ III, which contains the M289L mutation inaddition to the H204R, T306K, and F309N mutations, also has thisimprovement.

Specific Activity:

The specific activity for SUPERSCRIPT™ III is about 25% higher thanSUPERSCRIPT™ II. RT DNA polymerase unit activity was assayed with(rA)₆₃₀.p(dT)₁₂₋₁₈ at 37° C. and protein concentration was determined byBradford assay. Table 14 shows the RNA-directed DNA polymerase specificactivity of SUPERSCRIPT™ II and SUPERSCRIPT™ III using(rA)₆₃₀.p(dT)₁₂₋₁₈ at 37° C. for 10 min.

TABLE 14 DNA Polymerase Specific Activity Enzyme (units/mg)SUPERSCRIPT ™ II 330,000 SUPERSCRIPT ™ III 410,000

Mono- and Divalent Metal Reaction Optima:

Using primed CAT RNA as substrate, the Mg²⁺ and KCl concentration optimawere determined and are shown in FIGS. 10A and 10B. The DNA polymeraseassay for SUPERSCRIPT™ III RT was conducted at 37° C. or 50° C. for 10minutes under various concentrations of Mg²⁺ (FIG. 10A) or KCl (FIG.10B). SUPERSCRIPT™ II at 37° C. is included for comparison. The Mg²⁺concentration optima is at 3 mM at both 37° C. and 50° C. Though at 50°C. there appears to be a broader working range of 2-5 mM without muchdifference. For KCl, the optima is at 75 mM at both temperatures, butthe working range is from 25-125 mM without much difference. Again atthe higher temperature a broader range is allowed (25-200 mM). Sincethere is no change in optima between SUPERSCRIPT™ II and III, the 5×First Strand Buffer from SUPERSCRIPT™ II can be used as SUPERSCRIPT™III's reaction buffer.

TdT Activity:

TdT or non-template directed nucleotide addition to the 3′ primer end isa well known property of many polymerases including RTs (Chen, D. etal., Biotechniques 2001; 30(3):574-582). Using a DNA template (FIG.11A), SUPERSCRIPT™ II adds 1-3 additional nucleotides to the end of thetranscript. SUPERSCRIPT™ III however has a greatly reduced TdT and on aDNA template will not add any detectable nucleotides. On an RNA template(FIG. 11B), SUPERSCRIPT™ II adds 1-2 bases with 69% of the primerextended after 10 minutes and 90% extended after an hour. SUPERSCRIPT™III does have some TdT activity on RNA template, but it is reduced withabout 14% extended after 10 minutes and 50% extended after an hour at45° C. as shown in FIG. 11B. This TdT activity is biased with theaddition of dATP being strongly favored followed by dGTP, dCTP and dTTPfor both SUPERSCRIPT™ II and III. TdT activity is temperature and timedependant. The optimal temperature for SUPERSCRIPT™ III is 45° C. to 50°C. while SUPERSCRIPT™ II is at 45° C. (FIG. 11). To further minimize TdTactivity in any RT one must shorten the extension time or lower thetemperature.

Fidelity assays. The results of the fidelity assays are shown in Tables15 and 16.

TABLE 15 rpsL Fidelity Assay Mutated RT Temp Wt rpsL rpsL SUPERSCRIPT ™III 45° C. 3.9 × 10⁻⁴ 3.4 × 10⁻⁵ SUPERSCRIPT ™ II 45° C. 3.5 × 10⁻⁴ 3.1× 10⁻⁵ MMLV H+ 37° C. 3.9 × 10⁻⁴ NA

TABLE 16 lacZα Fidelity Assay RT Temp Error Rate SUPERSCRIPT ™ III 45°C. 2.9 × 10⁻⁵ 50° C. 2.7 × 10⁻⁵ 55° C. 1.8 × 10⁻⁵ SUPERSCRIPT ™ II 45°C. 2.9 × 10⁻⁵ MMLV H+ 45° C. 4.8 × 10⁻⁵

Fidelity assays for SUPERSCRIPT™ III, SUPERSCRIPT™ II and MMLV Rnase H+RTs. rpsL assay was conducted with either WT rpsL gene or rpsL gene withmutation hotspots removed. A wt rpsL DNA fragment was used as control tosee the spontaneously mutation. The error rate is about 10⁻⁶ to 10⁻⁷.The lacZα assay was conducted with gapped DNA starting substrate used asa control with a background error rate of 2.2×10⁻⁵.

Thermal Stability of Purified Mutant RTs: The rate of chemical RNAbreakdown catalyzed by Mg²⁺ increases dramatically as the temperature isincreased above 55° C. to 60° C. under cDNA synthesis reactionconditions. In order to characterize their intrinsic thermal stability,the half lives of M-MLV, SUPERSCRIPT™ II and SUPERSCRIPT™ III weremeasured at elevated temperatures. The RT enzymes were first diluted to0.2-0.5 Unit/μl in the 1× first strand buffer in the presence of 0.05%of NP-40. If diluted in absence of detergent, the RT activity is reducedto more than 90% without incubation (data not shown). FIGS. 12A, 12B,and 12C and Table 17 show the half-lives of M-MLV, SUPERSCRIPT™ II, andSUPERSCRIPT™ III at various temperatures. The RT half-lives weredetected in 1× First strand reaction buffer in presence of 0.05% NP-40.Mixtures (20 μl) were incubated for various times in 0.5-ml tubes in athermocycler at 50° C., 55° C., or 60° C. and contained 1× First StrandBuffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂), 10 mM DTT, 3 mMMgCl₂ (total to 6 mM), 0.5 mM dTTP, 0.05% (v/v) NP-40, [methyl-³H]dTTP(20 μCi/ml), and 5 μl of RT. Incubation was stopped by placing the tubein ice. An aliquot (5 μl) was assayed for remaining units with 5 mMpoly(A)/3 mM oligo(dT)₂₅. The half live of SUPERSCRIPT™ III at 50° C.was 220 minutes, about 35 fold longer than that of SUPERSCRIPT™ II. Thehalf live of SUPERSCRIPT™ III at 55° C. was 24 min, ˜10 fold longer thanthat of SUPERSCRIPT™ II.

TABLE 17 Summary of RT Half lives at 50° C., 55° C., and 60° C. M-MLVSUPERSCRIPT ™ II SUPERSCRIPT ™ III (min) (min) (min) 50° C. ND 6.1 22055° C. 1.5 2.2 24 60° C. 0.6 ND 2.5

Full-Length cDNA Profiling of RT:

A practical method for judging the useful higher temperature of the DNApolymerase activity of RT is an assessment of the effect of increasingreaction temperature on the amounts of full-length cDNA productssynthesized by RT from mixture of cRNA of various lengths. The labeledfull-length cDNA products can be separated and quantified on an alkalineagarose gel. As the reaction temperate is increased, full-length cDNAproducts disappear starting with those derived from longer cRNA until atemperature is reached where no discernible full-length product of anylength is synthesized. For comparison, we included Clontech'sPowerScript RT, Stratagene's StrataScript RT, Promega's ImProm II RT,and SUPERSCRIPT™ II (Invitrogen Corporation, Carlsbad, Calif., catalog#18064022). Only SUPERSCRIPT™ III could produce a clear 9.5 kbfull-length product at 50° C., and SUPERSCRIPT™ II can get a very faint9.5 kb band.

To compare RT from different companies, the same number of units of RTwere used for comparison. PowerScript RT is ˜350 unit/μl, StrataScriptRT is 50 unit/μl, ImProm II is 50 unit/μl. SUPERSCRIPT™ II,SUPERSCRIPTT™ III and PowerScript RT were diluted to 50 unit/μl in RTdilution buffer. 100 units (2 μl) of RT enzyme were used. The amounts offull-length products generated were determined and are shown in Tables17 and 18 for various length templates. SUPERSCRIPT™ III RT showsimproved performance at 50° C. and 55° C. compared to the other RTs.

TABLE 17 Comparison of full length cDNA produced by various RTs. TotalAmount of cDNA Full-Length cDNA Temp. RT Pmol 7.5 kb (ng) 2.4 kb (ng)45° C. PowerScript 293 75 64 StrataScript 199 47 35 ImProm II 256 41 44SUPERSCRIPT ™ III 345 99 72 SUPERSCRIPT ™ II 300 100 59 50° C.PowerScript 180 54 31 StrataScript 9 <2 <2 ImProm II 42 <2 <2SUPERSCRIPT ™ III 323 97 68 SUPERSCRIPT ™ II 170 46 22 55° C.PowerScript 5 <2 <2 StrataScript 5 <2 <2 ImProm II 3 <2 <2 SUPERSCRIPT ™III 37 3 22 SUPERSCRIPT ™ II 3 <2 <2

The amounts of full-length product were established by cutting theregion in a dried 1.2% alkaline agarose gel corresponding to the size ofeach full-length band and counting it in a scintillation counter. Onlyamounts of full-length products >2 ng could be seen as discernible bandson the gel autoradiograph. The cDNA synthesis reactions were performedin 20 μl with 0.25 μg of 2.4 kb, 0.5 mg of 7.5 kb cRNA, and 0.1 μg ofoligo (dT)₂₅. If provided, buffers and other components were used fromtheir own kits plus 0.1 μl of α-³²P-dCTP (10 μCi/μl). All the reactionswere “hot start.” In “hot start” reactions, tubes were incubated atreaction temperature for 2 minutes before adding enzyme. 2 μl of RT (50u/μl) was added and incubated at 45° C. or 50° C. or 55° C. for 50 min.The reactions were stopped by adding 10 μl of 0.5 M EDTA. After ethanolprecipitation, the products were loaded on 1.2% agarose gel. The driedgel was exposed on the film for 1 hour.

TABLE 18 Comparison of full length cDNA produced by various RTs. Amountof full length cDNA (ng) RT Temp (° C.) 9.5 kb 7.5 kb SUPERSCRIPT ™ 4519 54 III 50 26 78 55 13 53 StratScript 45 4 10 50 0 0 55 0 0 ImProm II45 3 9 50 0 0 55 0 0 OmniScript 45 0 0 50 0 0 55 0 0 PowerScript 45 1938 50 3 25 55 0 0 M-MLV 45 10 39 50 0 3 55 0 0 ThermoScript 45 16 38 5029 56 55 3 13

cDNA synthesis reaction mixtures contained 9.5 kb (1.1 μg) and 7.5 kb(0.8 μg) cRNA. The cDNA synthesis reactions were performed in 20 μl with20 μg of Hela total RNA, and 0.5 μg of oligo (dT)₂₅. If provided,buffers and other components used were from their own kits plus 0.1 μlof α-³²P-dCTP (10 μCi/μl). All the reactions were “hot start,” 1 μl ofRT was added and incubated at 45° C. or 55° C. for 50 min. 400 or 200units of SUPERSCRIPT™ III were used for comparison. The reactions werestopped by adding 10 μl of 0.5 M EDTA. After ethanol precipitation, theproducts were loaded on 1.2% agarose gel. The dried gel was exposed onthe film for 1 hour.

In an alternate procedure for comparing RTs from various sources, acomparison of cDNA synthesis from Hela total RNA was performed. The cDNAsize synthesized by SUPERSCRIPT™ III RT at 55° C. is from 0.5-10 kb.cDNA synthesis reactions were performed in 20 μl with 20 μg of Helatotal RNA, and 0.5 μg of oligo(dT)₂₅. If provided, buffers and othercomponents used were from their own kits plus 0.1 μl of α-³²P-dCTP (10μCi/μl). All the reactions were “hot start,” 1 μl of RT was added andincubated at 45° C. or 55° C. for 50 min. 400 or 200 units ofSUPERSCRIPT™ III were used for comparison. The reactions were stopped byadding 10 μl of 0.5 M EDTA. Total cDNA synthesis was obtained byTCA-precipitation of 5 μl of mixtures. After ethanol precipitation ofthe rest of the mixture, the products were loaded on 1.2% agarose gel.The dried gel was exposed on the film for 1 hour.

Each reaction was performed according to each vendor's recommendation.In these experiments, we used the buffers provided with the RTs. DTT,RNase inhibitor, dNTP were used at the recommended concentration andfrom the kit if provided. Otherwise, Invitrogen's reagents were used. 1μl of RT was used in these competitive audit. Table 18 shows the resultwhen using 9.5 kb and 7.5 kb cRNA. FIG. 13 shows the results of acompetitive audit for cDNA profiling using poly (A)-tailed RNA ladder.The cDNA synthesis reactions were performed in 20 μl with 2 μg of RNALadder and 0.5 μg of oligo(dT)₂₅. If provided, buffers and othercomponents were used from their own kits plus 0.1 μl of α-³²P-dCTP (10μCi/μl). All the reactions were “hot start,” 1 μl of RT was added andincubated at 45° C. or 50° C. or 55° C. for 50 min 400 or 200 units ofSUPERSCRIPT™ III were used for comparison. The reactions were stopped byadding 10 μl of 0.5 M EDTA. After ethanol precipitation, the productswere loaded in different order on two 1.2% agarose gels. The dried gelswere exposed on the film for 1 hour.

Example 8 RT-PCR Applications

RT-PCR has become an effective tool in such applications as detectingRNA, gene expression, profiling, gene quantitation, and cloningfull-length genes. An RNase H minus mutant of MMLV RT, SUPERSCRIPT™ IIRT, is widely accepted as the RT choice since it has already proven thatit produces higher cDNA yield and longer target capacity than MMLV RT.

Recently we have engineered a new generation RT-SUPERSCRIPT™ III™ RT. Inaddition to all the premier features of SUPERSCRIPT™ II RT, it alsoprovides a high temperature RT capacity up to 55 degrees, which isapproximately 5 degrees or more higher than SUPERSCRIPT™ II RT. Theenzyme half-life of SUPERSCRIPT™ II RT and SUPERSCRIPT™ III RT at 50° C.is 6 minutes and 220 minutes respectively. Efficient full-length cDNAsynthesis activity occurs with SUPERSCRIPT™ III even at 55 degrees RTtemperature. High yield and more specific RT-PCR products were alsoproduced by this enzyme at elevated RT temperatures. SUPERSCRIPT™ IIIprovides a tool for efficient cDNA synthesis for difficult RNA targetssuch as high GC and secondary structured templates.

The enzyme has also been optimized for RT-PCR for both sensitivity andlong targets. We have demonstrated that SUPERSCRIPT™ III RT is able toamplify as little as 1 pg of starting HeLa RNA and amplify targets up to12.3 kb in length. This enzyme, when used with the accompanying buffersand conditions, provides performance better than that of other MMLVderivative reverse transcriptases.

Materials and Methods

Kit Components:

SUPERSCRIPT™ III RT (200 U/μl), 5× First-Strand Buffer, 0.1M DTT.

SUPERSCRIPT™ III RT=H204R, M289L, T306K, and F309N mutations and may bereferred to as the LEFN RT.

5× First-Strand Buffer: 250 mM Tris-HCl (pH 8.3), 375 mM potassiumchloride, and 15 mM magnesium chloride.

SUPERSCRIPT™ III RT Storage Buffer: 20 mM Tris-HCl, pH 7.5, 100 mM NaCl,1 mM EDTA, 1 mM DTT, 0.01% NP-40, 50% (v/v) glycerol.

RNA: Total HeLa RNA and total rat brain RNA were isolated using TRIZOL®Reagent.

Gel Electrophoresis: RT-PCR products (10 μl) were analyzed byelectrophoresis on 0.8% to 1.5% (w/v) agarose gels in 0.5×TBE with 0.4μg/ml ethidium bromide.

RT-PCR Procedure: First-Strand cDNA Synthesis for RT-PCR: A 20-μlreaction volume can be used for 10 μg-5 μg of total RNA or 10 pg-500 ngof mRNA. Add the following components to a nuclease-free microfuge tube:1 μl Oligo(dT)₂₀ (500 μg/ml), 10 pg to 5 μg total RNA, 1 μl 10 mM dNTPmix (10 mM each dATP, dTTP, dGTP, and dCTP at neutral pH), and sterile,distilled water to 12 μl. Alternatively, 50-250 ng random primers or 2pmole of a gene specific primer may be used. Use of random primersrequires incubation at 25° C. for 10 min before the 50° C. incubation.

Heat the mixture at 65° C. for 5 min and then place on ice. Collect thecontents of the tube by brief centrifugation and add: 4 μl 5×First-Strand Buffer, 2 μl 0.1 M DTT, 1 μl RNaseOUT RecombinantRibonuclease Inhibitor (40 units/μl, when using less than 50 ng ofstarting RNA, the addition of an RNase inhibitor (e.g., RNaseOUT,Invitrogen Corporation, Carlsbad, Calif., catalog #10777019) isessential).

Mix the contents of the tube gently and incubate at 50° C. for 2minutes. Add 1 μl (200-400 units) of SUPERSCRIPT™ III RT, mix bypipetting gently up and down. Incubate 50 min at 50° C. (cDNA synthesiscan be performed at 42° C.-60° C. for oligo(dT)₂₀ or gene specificprimers). Inactivate the reaction by heating at 70° C. for 15 min. ThecDNA can now be used as a template for amplification in PCR or can bestored at −20° C. until use. However, amplification of some PCR targets(those >1 kb) may require removal of RNA complementary to the cDNA. Toremove RNA complementary to the cDNA, add 1 μl (2 units) of E. coliRNase H and incubate at 37° C. for 20 min.

RT-PCR Optimization

For the following experiments, either EFN (His-tag), EFN (no tag), LEFN(His-tag), or LEFN (tag, no His) were used in finding the optimalconditions for LEFN (tag, no His). These 4 reverse transcriptases differin their thermal-stability profiles. The modified version ofSUPERSCRIPT™ III, EFN, contains the H204R, T306K, and F309N mutations.LEFN, otherwise known as SUPERSCRIPT™ III RT, contains the M289Lmutation in addition to the H204R, T306K, and F309N mutations.

Unless otherwise noted, 20 μl RT reactions were typically done using 2.5μM Oligo(dT)₂₀ (500 ng Oligo(dT)₁₂₋₁₈ for SUPERSCRIPT™ II RT), 0.5 mMdNTPs, 1× First-Strand Buffer, 10 mM DTT, and 40 units of RNaseOUT. In“hot start” reactions, tubes were incubated at reaction temperature for2 minutes before adding enzyme. Reactions were treated with 2 units ofRNase H at 37° C. for 20 min after cDNA synthesis.

For the evaluation of these RTs in RT-PCR, standard 50 μl PCR reactionswere performed. For primer sets deigned to amplify<4 kb, PCR reactionsconsisted of 0.2 μM primers, 200 μM each dNTP, 1×PCR Buffer, 1.5 mMMgCl₂, 2 μl of the cDNA reaction, and 2 units of PLATINUM® Taq DNAPolymerase. For primer sets designed to amplify>4 kb, PCR reactionsconsisted of 0.2 μM primers, 200 μM each dNTP, 1× High Fidelity PCRBuffer, 2 mM MgSO₄, 2 μl of the cDNA reaction, and 1 unit of PLATINUM®Taq DNA Polymerase High Fidelity. After an incubation of 94° C. for 2min, amplification was 35 to 40 cycles of 94° C. for 15 s, 55° C.-60° C.for 30 s, and 68° C. for 1 min/kb. 10 μl of the PCR reactions wereanalyzed on agarose gels containing 0.4 μg/ml EtBr. Primers used in PCRare found in Table 19.

cDNA synthesis buffer: Reactions with 50 or 200 units of SUPERSCRIPT™ IIRT or EFN (His-tag) were assembled using the reagent systems ofSUPERSCRIPT™ II RT (stand-alone), SUPERSCRIPT™ H First-Strand SynthesisSystem for RT-PCR, and ThermoScript RT-PCR System. The conditions foreach are as follows:

-   -   1) SUPERSCRIPT™ II RT stand-alone: 50 mM Tris-HCl (pH 8.3), 75        mM KCl, 3 mM MgCl₂, 0.5 mM dNTPs    -   2) SUPERSCRIPT™ II First-Strand Synthesis System for RT-PCR: 20        mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl₂, 0.5 mM dNTPs    -   3) ThermoScript RT-PCR System: 50 mM Tris-acetate (pH 8.4), 75        mM potassium acetate, 8 mM magnesium acetate, 1 mM dNTPs        20 μl RT reactions containing 1 pg to 100 ng of starting total        HeLa RNA were performed at 45° C. for 50 min followed by 85° C.        for 5 min. 2 μl of the resulting cDNA were added to 50 μl PCR        reactions containing the β-actin 353 bp, BF 2.4 kb, Pol ε 6.8        kb, or APC 8.5 kb primer set (see Table 19).

Magnesium chloride was titrated into cDNA synthesis buffers in finalreaction concentrations of 1 to 10 mM, with dNTP concentrations of 0.5mM and 1 mM tested concurrently. These buffers were used in 20 μl RTreactions performed at 45° C. for 50 min followed by 85° C. for 5 minwith 1 pg, 100 ng, or 5 μg of total HeLa RNA and 50 units or 200 unitsof EFN (His-tag). 2 μl of the resulting cDNA were added to 50 μl PCRreactions containing the β-actin 353 bp or Pol ε 6.8 kb target (the 5 μgcDNA samples were diluted 100 fold before addition).

5× First-Strand Buffer containing Tris-HCl at pH 8.0, 8.4, 8.8 and 8.3were prepared to determine the ideal pH for the enzyme to function athigher reaction temperatures. 20 μl RT reactions containing 100 ng to500 ng of total HeLa RNA and 200 units of SUPERSCRIPT™ II or 400 unitsof LEFN (His-tag) were performed at 50° C.-60° C. for 50 min (hot start)followed by 70° C. for 15 min using these different buffers. 2 μl of theresulting cDNA were added to 50 μl PCR reactions containing the BF 2.4kb or Pol ε 6.8 kb primer set.

RT reaction temperature: SUPERSCRIPT™ II RT (50 units), EFN (His-tag)(50 and 200 units), and ThermoScript RT (15 units) were compared in RTreactions from 42° C. to 60° C. 20 μl RT reactions containing 1 pg to100 ng of starting total HeLa RNA were performed at the giventemperatures for 50 min (hot start) followed by 85° C. for 5 min. 2 μlof the resulting cDNA were added to 50 μl PCR reactions containing theβ-actin 353 bp, TSC 5.3 kb, or Pol ε 6.8 kb primer set.

SUPERSCRIPT™ II RT and EFN (no tag) were compared at 200 units in RTreactions at 45° C. and 50° C. 20 μl RT reactions containing 1 ng to 1μg of total HeLa RNA were performed at the given temperatures for 50 min(hot start) followed by 70° C. for 15 min. 2 μl of the resulting cDNAwere added to 50 μl PCR reactions containing primer sets from 2.4 kb to9.3 kb.

SUPERSCRIPT™ II RT, EFN (His-tag), and EFN (no tag) were compared at 200units in RT reactions from 45° C. to 55° C. 20 μl RT reactionscontaining 1 pg to 100 ng of total HeLa RNA were performed at the giventemperatures for 50 min (hot start) followed by 70° C. for 15 min. 2 μlof the resulting cDNA were added to 50 μl PCR reactions containingeither the β-actin 353 bp or Pol ε 6.8 kb primer set.

SUPERSCRIPT™ II RT, EFN (His-tag), EFN (no tag), and LEFN (His-tag) werecompared at 200 units in RT reactions from 45° C. to 55° C. 20 μl RTreactions containing 1 pg to 1 μg of total HeLa RNA were performed atthe given temperatures for 50 min (hot start) followed by 70° C. for 15min. 2 μl of the resulting cDNA were added to 50 μl PCR reactionscontaining either the β-actin 353 bp or Pol ε 6.8 kb primer set.

SUPERSCRIPT™ II RT, EFN (His-tag), EFN (no tag), and LEFN (His-tag) werecompared at 200 units in RT reactions from 45° C. to 55° C. with agene-specific primer (CBP 1.6 kb) instead of Oligo(dT). 20 μl RTreactions containing 1 ng or 10 ng of total HeLa RNA were performed atthe given temperatures for 50 min (hot start) followed by 70° C. for 15min. 2 μl of the resulting cDNA were added to 50 μl PCR reactionscontaining the CBP 1.6 kb primer set.

SUPERSCRIPT™ II RT, EFN (no tag), EFN (His-tag), and LEFN (His-tag) werecompared at 200 and 400 units in RT reactions from 45° C. to 60° C. 20μl RT reactions containing 500 ng of total HeLa RNA were performed atthe given temperatures for 50 min (hot start) followed by 70° C. for 15min. 2 μl of the resulting cDNA were added to 50 μl PCR reactionscontaining either the BF 2.4 kb or Pol ε 6.8 kb primer set.

SUPERSCRIPT™ II RT, EFN (His-tag), LEFN (His-tag), and LEFN (tag, noHis) were compared at 200 and 400 units in RT reactions from 45° C. to60° C. 20 μl RT reactions containing 500 ng of total HeLa RNA wereperformed at the given temperatures for 50 min (hot start) followed by70° C. for 15 min. 2 μl of the resulting cDNA were added to 50 μl PCRreactions containing either the BF 2.4 kb or Pol ε 6.8 kb primer set.

RT enzyme concentration: 20 μl RT reactions containing 0.1 pg to 1 μg ofstarting total HeLa RNA and 25 units to 250 units of EFN (His-tag) wereperformed at 45° C. for 50 min followed by 85° C. for 5 min. 2 μl of theresulting cDNA were added to 50 μl PCR reactions containing either theβ-actin 353 bp, CBP 1.6 kb, TSC 5.3 kb, Pol ε 6.8 kb, or APC 8.5 kbprimer set.

SUPERSCRIPT™ II RT and LEFN (His-tag) were compared at 200 units and 400units in RT reactions from 50° C. to 60° C. 20 μl RT reactionscontaining 10 ng to 1 μg of total HeLa RNA were performed at the giventemperatures for 50 min (hot start) followed by 70° C. for 15 min. 2 μlof the resulting cDNA were added to 50 μl PCR reactions containingeither BF 2.4 kb or Pol ε 6.8 kb primer set.

SUPERSCRIPT™ II RT and LEFN (His-tag) were compared at 200 units and 400units (LEFN was also used at 800 units) in RT reactions (with or without0.05% Triton X-100) from 50° C. to 60° C. 20 μl RT reactions containing500 ng of total HeLa RNA were performed at the given temperatures for 50min (hot start) followed by 70° C. for 15 min. 2 μl of the resultingcDNA were added to 50 μl PCR reactions containing either BF 2.4 kb orPol ε 6.8 kb primer set. Triton did not improve the yield of thereaction and actually reduced the yield slightly.

LEFN (tag, no His) was compared at 50, 200, and 400 units to determinethe effect on sensitivity. 20 μl RT reactions containing 0.1 μg to 100pg of total HeLa RNA were performed at 50° C. under both hot start andcold start conditions followed by 70° C. for 15 min. 2 μl of theresulting cDNA were added to 50 μl PCR reactions containing either theβ-actin 353 bp or GAPDH 532 bp primer set.

Comparison of 85° C., 5 min and 70° C., 15 min Inactivation Steps.SUPERSCRIPT™ II RT and EFN (no tag) were compared in RT reactions witheither a 70° C., 15 min or an 85°, 5 min inactivation step. 20 μl RTreactions containing 1 pg to 100 ng of starting total HeLa RNA and 200units of RT were performed at 45° C. for 50 min followed by one of theinactivation steps. 2 μl of the resulting cDNA were added to 50 μl PCRreactions containing either the β-actin 353 bp, CBP 1.6 kb, Pol ε 3.5kb, TSC 5.3 kb, or Pol ε 6.8 kb primer set.

Priming with Oligo(dT)₂₀, Random Hexamers, and Gene-Specific Primers(GSP). EFN (no tag) and SUPERSCRIPTT™ II RT were used in RT reactionscontaining either 2.5 μM oligo(dT)₂₀, 50 ng random hexamers, or 0.1 μMGSP. 20 μl RT reactions containing 1 ng to 1 μg of starting total HeLaRNA and 200 units of RT were performed at 45° C. for 50 min followed by70° C. for 15 min. 2 μl of the resulting cDNA were added to 50 μl PCRreactions containing either the CBP 1.6 kb, TSC 5.3 kb, or APC 8.5 kbprimer set (FIG. 21).

Comparison of reverse transcriptases from various sources. LEFN (tag, noHis) was compared to Clontech PowerScript™ RT, Stratagene StrataScript™RT, Qiagen SensiScript™ RT, Qiagen OmiScript™ RT, and Promega ImProm-II™RT System. RT reactions containing 0.1 pg to 1 μg of starting total HeLaRNA or 1 μg of rat brain RNA were performed in duplicate with each ofthe reverse transcriptases using the procedure and conditionsrecommended by each supplier. 10% of the cDNA reactions were added to 50μl PCR reactions containing either the β-actin 353 bp, GAPDH 532 bp, CBP1.6 kb, BF 2.4 kb, VIN 4.6 kb, FIB 9.4 kb, or Dynein 12.3 kb primer set.

TABLE 19 Primer list Human β-actin-353 bp senseGCTCGTCGTCGACAACGGCTC (SEQ ID NO: 12) antisenseCAAACATGATCTGGGTCATCTTCTC (SEQ ID NO: 13) Human GAPDH-532 bp senseGTGAAGGTCGGAGTCAACGGATTT (SEQ ID NO: 14) antisenseCACAGTCTTCTGGGTGGCAGTGAT (SEQ ID NO: 15)Human Cap Binding Protein (CBP)-1.6 kb senseATGGCGATCGTCGAACCGGA (SEQ ID NO: 16) antisenseCACTGTCTTAATATGAATGGGACCTACTGAG (SEQ ID NO: 17)Human B-factor properdin (BF)-2.4 kb senseGAGCCAAGCAGACAAGCAAAGCAAGC (SEQ ID NO: 18) antisenseTGTTTTAATTCAATCCCACGCCCCTGT (SEQ ID NO: 19) Human DNA Polymerase ε(Pol ε)-3.5 kb sense AAGGCTGGCGGATTACTGCC (SEQ ID NO: 20) antisenseGATGCTGCTGGTGATGTACTC (SEQ ID NO: 21) Human Vinculin (VIN)-4.6 kb senseGAGGAGGGCGAGGTGGACGGC (SEQ ID NO: 22) antisenseGAACTAACACACAGCGATGGGTGGGAA (SEQ ID NO: 23)Human Tuberous Sclerosis 2 (TSC-2)-5.3 kb senseGGAGTTTATCATCACCGCGGAAATACTGAGAG (SEQ ID NO: 24) antisenseTATTTCACTGACAGGCAATACCGTCCAAGG (SEQ ID NO: 25)Human DNA Polymerase s (Pol ε)-6.8 kb senseCGCCAAATTTCTCCCCTGAA (SEQ ID NO: 26) antisenseCCGTAGTGCTGGGCAATGTTC (SEQ ID NO: 27)Human Adenomatous Polyposis coli (APC)-8.5 kb senseGCTGCAGCTTCATATGATCAGTTGTTA (SEQ ID NO: 28) antisenseAATGGCGCTTAGGACTTTGG (SEQ ID NO: 29) Human Fibrillin (FIB)-9.4 kb senseTGGAGGCTGGGAACGTGAAGGAAA (SEQ ID NO: 30) antisenseACAGGAATGACCGAGGGTAATCTTGGC (SEQ ID NO: 31) Rat Dynein-12.3 kbsense GCGGCGCTGGAGGAGAA (SEQ ID NO: 32)antisense AGGTGGCGGCTCAAACACAAAG (SEQ ID NO: 33)H-fibroblast growth factor 11 (FGF)-240 bp;  97° C. denaturing, 60°C. annealing temp.sense (f1-60)-CGGGTGGTAACTGGCTGCTGTGGA (SEQ ID NO: 34)antisense TGGAGGCTGGGAACGTGAAGGAAA (SEQ ID NO: 35)antisense (r2-299)-GCGGACCTCCCGCTTCTGCCGGA (SEQ ID NO: 36)H-cystathionine-beta-synthase (CBS 2.4)-2390 bp;  64°C. annealing temperaturesense(f2-71)-CCAAGTAAAACAGCATCGGAACACCAGG (SEQ ID NO: 37)antisense (r2-2460)-AAAGTCGATCAGCAGTTGCCAGGGG (SEQ ID NO: 38)H-topoisomerase I (TOP3.2)-3162 bp;  60° C. annealing temperaturesense (f2-80)-CCCACAGTCACCGCCGCTTACCT (SEQ ID NO: 39)antisense (r1-3241)-CTTCATCCCTCCCCAACCCCAATCT (SEQ ID NO: 40)H-vinculin (VIN4.6)-4641 bp;  66° C. annealing temperaturesense (f1-132)-GAGGAGGGCGAGGTGGACGGC (SEQ ID NO: 41)antisense (r2-4772)-GAACTAACACACAGCGATGGGTGGGAA (SEQ ID NO: 42)H-PolE (PolE 6.8)-6800 bp;  60° C. annealing temperaturesense-CGCCAAATTTCTCCCCTGAA (SEQ ID NO: 43)antisense-CCGTAGTGCTGGGCAATGTTC (SEQ ID NO: 44)H-β-actin (13act353)-353 bp;  55° C. annealing temperature sense-5′GCTCGTCGTCGACAACGGCTC (SEQ ID NO: 45)antisense-ACCACATGATCTGGGTCATCTTCTC (SEQ ID NO: 46)Comparison of SUPERSCRIPT™ III to SUPERSCRIPT™ II

A comparison of SUPERSCRIPT™ III to SUPERSCRIPT™ II was made using thefollowing assay. Total HeLa RNA (1 μg) was combined with 10 pmol genespecific primer (antisense primer, Table 19) and hybridized at 65 C for5 min. Transcription with 200 U of each enzyme was carried out at 42°C., 50° C., or 55° C. for 50 min. Reverse transcription for comparisonof enzyme preparations were performed using 1 pg, 10 pg, 100 ng or 1 μgof total RNA from HeLa cells with 50 pmol OligodT₍₂₀₎ at 55° C. 50 min.Reaction components included 0.5 mM dNTPs, 0.01 M DTT, 40 U RNase OUT™,50 mM Tris-KCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂. After reversetranscription, the enzyme was heat inactivated by incubation at 70° C.for 15 min. After a 20 min. incubation with 2 U RnaseH at 37° C., 2 μLof the 20 μL reaction was amplified by PCR using Platinum Taqhi-fidelity. The amplification reaction contained 60 mM Tris-SO₄ (pH8.9), 18 mM NH₄SO₄, 2 mM MgSO₄, 0.2 mM each dNTP, 0.2 μM each primer(Table 19), and 1 U Platinum Taq high fidelity. The amplification wascarried out by an initial 94° C. denaturation step for two min.,followed by 35 cycles of the following steps: 94° C. 15 sec., X° C. 30sec., 68° C. 1 min/kb, where X indicates the annealing temperature ofthe primer set listed in Table 19 (55° C.-66° C.). High G/C contentproducts were amplified with the following cycling beginning with a 94°C. denaturation step for two min. followed by 35 cycles of thefollowing: 97° C., 15 sec.; 60° C. 30 sec.; 68° C. one min. Thereactions were combined with 5 μL 10× BlueJuice® and 20% (11 μL) of eachreaction was electrophorsed through 0.8% or 1.5% agarose gels stainedwith 0.5 μg/ml ethidium bromide.

Results

RT-PCR Optimization: cDNA synthesis buffer. SUPERSCRIPT™ III RT was usedwith the SUPERSCRIPT™ II RT stand-alone conditions. These conditionswere found to be optimal for SUPERSCRIPT™ III RT in first-strandsynthesis. No significant difference in performance in RT-PCR was seenwith SUPERSCRIPT™ III RT using the reaction conditions of SUPERSCRIPT™II RT stand-alone, SUPERSCRIPT™ II First-Strand Synthesis System forRT-PCR, or ThermoScript RT-PCR System with a β-actin 353 bp target orwith larger targets.

A magnesium concentration of 3 mM was chosen for SUPERSCRIPT™ III RT asthis the minimum concentration needed in the RT reaction and it was thesame concentration found with the SUPERSCRIPT™ II RT stand-alone. With0.5 mM dNTPs, optimal performance was seen with magnesium concentrationsfrom 3 mM to 7 mM with both low RNA concentrations of 1 pg, 100 ng, and5 μg.

The pH of the 5× First-Strand Buffer will remain at 8.3 as this is thepH found in the SUPERSCRIPT™ II RT buffer and little difference was seenwith different pHs at higher temperatures (FIG. 14).

RT reaction temperature. 200 units of LEFN(SUPERSCRIPT™ III RT) showedlittle reduction in product up to 50° C., which is 5° C. higher than thecapabilities of SUPERSCRIPT™ II RT (50 U), and 5° C. lower than that ofThermoScript RT (15 U). Results were similar with both low levels of RNAand the β-actin 353 bp primer set, and higher levels of RNA and the TSC5.3 kb primer set.

When a His-tag is part of the RT, slightly higher thermal-stability isnoted. RT reactions containing 10 to 1000 ng of total HeLa RNA and 200units of SUPERSCRIPT™ II or LEFN RT were run at 45° C. to 55° C. for 50minutes in a hot start reaction. 2 μl of the resulting cDNA productswere then added to 50 μl PCR reactions containing the Pol ε 6.8 kbprimer set. Resulting PCR products were then run on a 0.8% agarose gelcontaining 0.4 mg/ml ethidium bromide. Not much difference was seenbetween 200 units of EFN (His-tag), EFN (no tag), and LEFN (His-tag)with the β-actin 353 bp primer set with all three showing product up to55° C. However, with the Pol ε 6.8 kb target, product was also seen upto 55° C. with all three mutants, except EFN (no tag) showed lessproduct at this temperature than either of the mutants with His-tags.

The same temperature profile as found with oligo(dT)₂₀ and the advantageof the His-tag was also seen with a gene-specific primer. RT reactionscontaining 1 to 10 ng of total HeLa RNA and 200 units of SUPERSCRIPT™II, EFN, or LEFN RT were run at 45° C. to 65° C. for 50 minutes in a hotstart reaction. 2 μl of the resulting cDNA products were then added to50 μl PCR reactions containing the CBP 1.6 kb primer set. Resulting PCRproducts were then run on a 0.8% agarose gel containing 0.4 mg/mlethidium bromide. Using a gene-specific primer in the RT reaction, 200units of EFN (His-tag), EFN (no tag), and LEFN (His-tag) all stillshowed CBP 1.6 kb product up to 55° C., but EFN (no tag) showed slightlyless than the others at 55° C.

In order to exploit the stability provided by the His-tag, LEFN with atag minus the 6×His was designed. RT reactions containing 500 ng oftotal HeLa RNA and 200 or 400 units of SUPERSCRIPT™ II, EFN, or LEFN RTwere run at 45° C. to 65° C. for 50 minutes in a hot start reaction. 2μl of the resulting cDNA products were then added to 50 μl PCR reactionscontaining the BF 2.4 kb primer set. Resulting PCR products were thenrun on a 0.8% agarose gel containing 0.4 mg/ml ethidium bromide. Littledifference was seen with the mutant when compared to both EFN (His-tag)and LEFN (His-tag), product was seen up to 60° C. with all three withthe BF 2.4 kb primer set (FIG. 15).

RT enzyme concentration. An experiment comparing units of EFN (His-tag)from 25 units to 250 units showed little difference in performance overthe entire range, when looking at both sensitivity with the β-actin 353primer set or with longer targets. RT reactions containing 0.1 to 1000ng of total HeLa RNA and 25 to 250 units of EFN were run at 45° C. for50 minutes in a hot start reaction. 2 μl of the resulting cDNA productswere then added to 50 μl PCR reactions containing the β-actin 353 bp,CBP 1.6 kb, TSC 5.3 kb, Pol ε 6.8 kb, or APC 8.5 kb primer set.Resulting PCR products were then run on a 0.8% agarose gel containing0.4 mg/ml ethidium bromide.

200 and 400 units of LEFN (tag, no His) showed slightly betterperformance than 50 units with low concentrations of RNA. RT reactionscontaining 0.1 to 100 pg of total HeLa RNA and 50 to 400 units of LEFNRT were run at 50° C. for 50 minutes in a hot start reaction. 2 μl ofthe resulting cDNA products were then added to 50 μl PCR reactionscontaining the β-actin 353 bp primer (not shown) or the GAPDH 532 primerset. Resulting PCR products were then run on a 1.5% agarose gelcontaining 0.4 mg/ml ethidium bromide. The higher RT units yieldedslightly more PCR product under hot start conditions with both β-actin353 bp (data not shown) and GAPDH 532 bp (FIG. 16).

Priming with Oligo(dT)₂₀, Random Hexamers, and Gene-Specific Primers(GSP). SUPERSCRIPT™ III RT performed similarly to SUPERSCRIPT™ II RTwhen RT reactions were run with different priming methods. Oligo(dT)yielded the cleanest product and highest specific yield with GSP havinghigh levels on non-specific products (at 55 degrees RT temperature) andrandom hexamers having high specificity but low yield.

Comparison of reverse transcriptases from various sources. Using β-actin353 bp primer set, SUPERSCRIPT™ III RT was again able to detect down to1 pg of starting HeLa RNA. RT reactions containing 1 to 100 pg of totalHeLa RNA were run with each RT using the reagents and conditionsspecified in each protocol. 2 μl of the resulting cDNA products werethen added to 50 μl PCR reactions containing the β-actin 353 bp primerset. Resulting PCR products were then run on a 1.5% agarose gelcontaining 0.4 mg/ml ethidium bromide. ImProm II RT and SensiScript werealso able to detect down to 1 pg, but StrataScript and OmniScript couldonly detect down to 10 pg, and PowerScript was unable to even detect 100pg of starting HeLa RNA.

With larger targets and higher concentrations of total HeLa RNA,SUPERSCRIPT™ III RT (LEFN tag, no His) performed significantly betterthan most of the competitors in relation to the RT-PCR product yield andlength. RT reactions containing 10 to 1000 ng of total HeLa RNA were runwith each RT using the reagents and conditions specified in eachprotocol. 2 μl of the resulting cDNA products were then added to 50 μlPCR reactions containing the CBP 1.6 kb, BF 2.4 kb, VIN 4.6 kb or 9.4 kbprimer set. Resulting PCR products were then run on a 0.8% agarose gelcontaining 0.4 mg/ml ethidium bromide. CLONTECH PowerScript only showedproduct when the total HeLa RNA was 1000 ng, and the yield of specificproducts was not as high as with SUPERSCRIPT™ III RT. Qiagen SensiScriptand OmniScript RT showed sufficient product yield with the smallertargets (CBP 1.6 kb and BF 2.4 kb), but were not able to detect thelonger targets. Stratagene StrataScript was able to detect all thetargets but with lower yields than SUPERSCRIPT™ III RT. PromegaImProm-II RT System showed performance similar to SUPERSCRIPT™ III RT,but did not have as high a yield with the longest target (FIB 9.4 kb).

Additional reactions with lower starting HeLa RNA concentrations showeda similar pattern. RT reactions containing 10 to 100 ng of total HeLaRNA were run with each RT using the reagents and conditions specified ineach protocol. 2 μl of the resulting cDNA products were then added to 50μl PCR reactions containing the CBP 1.6 kb, BF 2.4 kb, VIN 4.6 kb or 9.4kb primer set. Resulting PCR products were then run on a 0.8% agarosegel containing 0.4 mg/ml ethidium bromide. CLONTECH PowerScript wasunable to detect any of the targets with 100 ng or less of startingtotal HeLa RNA. Qiagen SensiScript was able to detect the VIN 4.6 kbtarget with this lower concentration of RNA when it had not been able todetect this target previously with 1 μg of starting total HeLa RNA.Promega ImProm-II did not perform as well as SUPERSCRIPT™ III RT withlower concentrations of RNA. SUPERSCRIPT™ III RT was the only RT thatwas able to detect the FIB 9.4 kb target with 100 ng starting total HeLaRNA.

RT-PCR analysis. SUPERSCRIPT™ II and SUPERSCRIPT™ III performance in hotstart RT-PCR amplification at 42° C., 50° C., pr 55° C. were comparedand the results are shown in FIG. 17 SUPERSCRIPT™ II (Panel A) orSUPERSCRIPT™ III (Panel B). Lanes (in duplicate) 1, 4, 7, and 10 areproducts reverse transcribed at 42° C. Lanes 2, 5, 8, and 11 areproducts reverse transcribed at 50° C. Lanes 3, 6, 9, and 12 areproducts transcribed at 55° C. Lanes 1-3 are the result of RNAs reversetranscribed by gene-specific priming from FGF, lanes 4-6 CBS 2.4, lanes7-9 from TOP 3.2, lanes 10-12 VIN 4.6. Arrows indicate expected productsizes of 240 bp, 2390 bp, 3162 bp, and 4641 bp. SUPERSCRIPT™ IItranscribed more robustly at 50° C. whereas SUPERSCRIPT™ III besttranscribes at 55° C. Amplification at 55° C. for some applications thatrequire higher annealing temperatures for gene specific priming and/orto remove secondary structure from an RNA template may be performed withthe polypeptides of the invention. This will allow the reduction ofnon-specific priming during reverse transcription with gene specificprimers and/or the increased reverse transcription of refractorytemplates.

SUPERSCRIPT™ III RT: Two different purification techniques. SUPERSCRIPT™III has been purified by two different methods (see above). In brief,Method A is similar to the purification technique used for SUPERSCRIPT™II, Method B differs from method A by the use of a membranepermeabilization protocol and filtration protocol to reduce cellulardebris which results in a high-purity preparation. RT-PCR was performedusing 200 U of SUPERSCRIPT™ III purified by Method A or Method B. RT-PCRwas performed using the Pol ε 6.8 kb primers and β-act 353 bp primers.Product yield and quality were compared for both purification methods.The methods yield similar results and either are viable methods forpurification of SUPERSCRIPT™ III.

Optimum SUPERSCRIPT™ III RT enzyme concentration. RT reactionscontaining 0.1 pg to 1000 ng of total HeLa RNA and 25 to 250 units ofSUPERSCRIPT™ II, EFN, or LEFN RT were run at 45° C. for 50 min (hotstart). 2 μl of the resulting cDNA were then added to 50 μl PCRreactions containing the β-actin 353 bp, CBP 1.6 kb, TSC 5.3 kb, Pol E6.8 kb, or APC 8.5 kb primer set. Resulting PCR products were then runon a 0.8% or 1.5% agarose gel containing 0.4 μg/ml ethidium bromide. Nosignificant differences was obtained from 25 units to 250 units ofSUPERSCRIPT™ III RT. (FIG. 18). However, higher amount of SUPERSCRIPT™III RT (400 units) shows higher RT-PCR product yield at an elevated RTtemperature (55 degrees, FIG. 19). In FIG. 19, RT reactions containing10 to 1000 ng of total HeLa RNA and 200 or 400 units of SUPERSCRIPT™ IIor LEFN RT were run at 50° C. to 60° C. for 50 min (hot start). 2 μl ofthe resulting cDNA were then added to 50 μl PCR reactions containing thePol ε 6.8 kb primer set. Resulting PCR products were then nm on a 0.8%agarose gel containing 0.4 μg/ml ethidium bromide. Unlike SUPERSCRIPT™II RT, increased amount of SUPERSCRIPT™ III RT (up to 400 units) did notinhibit subsequent PCR amplification (FIG. 20). In FIG. 20, RT reactionscontaining 0.1 to 100 pg of total HeLa RNA and 50 to 800 units of LEFNRT were run at 50° C. for 50 min in hot start conditions (dT)₂₀). 2 μlof the resulting cDNA were then added to 50 μl PCR reactions containingthe GADPH 532 bp primer set. Resulting PCR products were then run on a0.8% agarose gel containing 0.4 μg/ml ethidium bromide.

SUPERSCRIPT™ III RT has been engineered to increase the temperature atwhich the enzyme can perform RT activity. The enzyme has been optimizedfor RT-PCR for sensitivity, yield, and target length. With thisoptimized system, it was demonstrated that SUPERSCRIPT™ III RT was ableto detect mRNA targets with as little as 1 pg of starting total HeLa RNAand to produce high yield cDNAs from targets up to 12.3 kb in size. Ithas the ability to function at elevated temperatures up to 55° C. and todetect a wide variety of targets. The performance of this enzyme issuperior to any other RTs commercially available, and the optimizedbuffers and RT protocol provide a sensitive and robust RNA detectionsystem.

Example 9 Use of Polypeptides of the Invention to Prepare LabeledNucleic Acid Molecules

Polypeptides of the invention can be used to prepare labeled nucleicacids from a variety of templates (e.g., total RNA, mRNA, etc.). Fordirect labeling using a polyA tailed RNA template, suitable reactionconditions may entail the use of from about 1 μg to about 1000 μg, fromabout 1 μg to about 500 μg, from about 1 μg to about 250 μg, from about1 μg to about 100 μg, from about 10 μg to about 1000 μg, from about 10μg to about 500 μg, from about 10 μg to about 250 μg, or from about 10μg to about 100 μg of RNA.

RNA can be mixed with a suitable primer (e.g., an oligo dT primer or agene specific primer). After mixing, the template primer mixture can beincubated at a suitable temperature (e.g., 70° C. for an oligo(dT)₂₅primer) and incubated for a suitable period of time (e.g., 5 minutes).Those skilled in the art can readily determine incubation times andtemperatures for any template primer pair using routine experimentation.The mixture may then be chilled on ice and the remaining reactioncomponents added.

Suitable reaction components, which may be provided in a reactionmixture and/or solution either individually or in combination, include abuffering agent, reducing agent, one or more nucleotides, at least oneof which may contain a label (e.g., a fluorescent label), one or morepolypeptide of the invention, and suitable diluent (e.g., H₂O). Asuitable reaction mixture can be prepared by combining 4 μl 5× firststrand buffer, 2 μl 0.1M DTT, 1 μl 10 mM dNTP, 2 μl 1 mM FluorescentdNTP, 2 μl SUPERSCRIPT™ III (200 u/μl), an aliquot containing RNA, anddH₂O to 20 μl. Additional reaction mixtures and/or solutions, as well ascomponents thereof, which may be used with this aspect of the inventionare described elsewhere herein. The mixture may be incubated at asuitable temperature, for example, from about 42° C. to about 60° C.,from about 45° C. to about 60° C., from about 48° C. to about 60° C.,from about 50° C. to about 60° C., from about 52° C. to about 60° C.,from about 55° C. to about 60° C., from about 42° C. to about 55° C.,from about 45° C. to about 55° C., from about 45° C. to about 50° C.,from about 45° C. to about 48° C., from about 48° C. to about 60° C.,from about 48° C. to about 55° C., from about 48° C. to about 52° C.,from about 50° C. to about 60° C., from about 50° C. to about 55° C., orfrom about 50° C. to about 52° C. The mixture may be incubated until asufficient incorporation of label is seen, for example, from about 5minutes to about 24 hours, from about 10 minutes to about 24 hours, fromabout 30 minutes to about 24 hours, from about 1 hour to about 24 hours,from about 2 hours to about 24 hours, from about 4 hours to about 24hours, from about 8 hours to about 24 hours, from about 30 minutes toabout 16 hours, from about 30 minutes to about 8 hours, from about 30minutes to about 4 hours, from about 30 minutes to about 2 hours, fromabout 30 minutes to about 1 hour, from about 1 hour to about 4 hours, orfrom about 1 hour to about 2 hours.

The reaction may be stopped, for example, by addition of a suitablestopping reagent (e.g., 5 μl of 0.5M EDTA in a 20 μl reaction). Theresultant labeled nucleic acids may be purified using any standardtechnique (e.g., column purification using a SNAP column, InvitrogenCorporation, Carlsbad, Calif.).

Labeled nucleic acid produced by the methods of the invention may beused for a variety of purposes, for example, to detect a targetsequence, which may be present on an array. Typically, labeled nucleicacid of the invention may be hybridized to one or more target sequences(e.g., a microarray).

In some embodiments, polypeptides of the invention may be used toprepare labeled nucleic acids by indirect labeling. For example, amodified nucleotide (e.g., an amino allyl nucleotide) may beincorporated into a nucleic acid molecule using the polypeptides of theinvention. The nucleic acid molecules containing the modified nucleotidemay then be reacted with a reactive molecule that comprises a detectablemoiety (e.g., a fluorescent moiety, radiolabel and the like). All or aportion of the reactive molecule and the detectable moiety may thenbecome attached to the nucleic acid molecule.

Suitable reaction conditions for indirect labeling include those listedabove. For example, RNA (e.g., 5-50 μg) can be mixed with a suitableprimer (e.g., an oligo(dT)₂₅ for polyA tailed RNA), mixed and incubated,for example, at 70° C. for 5 min, and then chilled on ice. To theprimer: template mixture, addition reaction components may be added. Forexample, for a 30 μl reaction volume, suitable component may include: 6μl 5× first strand buffer, 1.5 μl 0.1M DTT, 1 μl RNaseOUT (40 u/μl), 1.5μl 10 mM dNTP mixture containing 10 mM dATP, 10 mM dCTP, 10 mM dGTP, 4mM dTTP, and 6 mM Aminoallyl-dUTP), 2 μl SUPERSCRIPT™ III (200 u/μl), Xμl dH₂O to a total volume of 30 μl. The mixture may be incubated at asuitable temperature for a suitable time such as those described above,for example, at 50° C. for 2 hours. The mixture may be treated todegrade the RNA template (e.g., by the addition of 15 μl 1N NaOH, andheating to 70° C. for 10 min). The pH of the solution may then beadjusted (e.g., by the addition of 15 μl 1N HCl) and the nucleic acidcontaining the modified nucleotide may be purified by standardtechniques (e.g., using a SNAP column, Invitrogen Corporation, Carlsbad,Calif.). The purified nucleic acid may be concentrated (e.g., by ethanolprecipitation).

After ethanol precipitation, the nucleic acid containing the modifiednucleotide may be resuspended in a buffer suitable for coupling thereactive molecule containing the detectable moiety to the nucleic acid.For example, the nucleic acid may be resuspended in 5 μl coupling buffer(e.g., 0.1 M sodium borate, pH 8.5). The reactive molecule (e.g., amolecule comprising a dye and a reactive functionality) may be added(e.g., 5 μl monofunctional Cy3 or Cy5 dye from APB Cat#PA23001 and #PA25001). When the reactive molecule is a dye, the dye may be suspendedin any suitable solvent (e.g., DMSO). In one embodiment, 1 pack of CyDyewas resuspended in 45 μl DMSO and used as above. The reaction may beincubated for a suitable length of time and at a suitable temperature,for example, at room temperature for 1 hour. When the dye is lightsensitive, the incubation is preferably performed in the dark. Thecoupling reaction is stopped by the addition of a reagent such as 5 μlof 4M hydroxylamine. The labeled nucleic acid is then purified (e.g.,using a SNAP column, Invitrogen Corporation, Carlsbad, Calif.).

Labeled nucleic acids (whether prepared by direct or indirect labeling)may be hybridized to one or more target sequences, which may be in anysuitable form, for example, in a microarray. The labeled nucleic acidsare denatured (e.g., by heating at 95° C. for 2 min). The denatured,labeled nucleic acids are brought into contact with the target sequencesin a suitable buffer. For example, a microarray slide can be placed in ahybridization buffer (e.g., 25% Formamide, 5×SSC, 50 mM MES pH 6.5, 0.1%SDS) that contains labeled nucleic acid (e.g., ˜1 μg of Dye-labelprobe). The microarray can be incubated for a suitable time at asuitable temperature, for example, at 42° C. for 16 hours. Themicroarray may then be washed one or more times, for example, thehybridization solution may be removed and the microarray washed in WashI (5×SSC, 0.2% SDS), followed by a wash in Wash II (1×SSC, 0.2% SDS) at42° C. for 5 min, followed by a wash in Wash III (0.1×SSC) for 2 min.The microarray may be dried, for example, by spin drying the microarrayat 600 rpm for 5 min.

The hybridized microarray may be analyzed using any techniques known inthe art.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporate by reference.

What is claimed is:
 1. A method for making one or more nucleic acidmolecules comprising mixing one or more nucleic acid templates with oneor more polypeptides having reverse transcriptase activity wherein thereverse transcriptase is an M-MLV reverse transcriptase comprising aminoacids 34-710 of SEQ ID NO: 2 and incubating the mixture under conditionssufficient to synthesize one or more first nucleic acid moleculescomplementary to all or at least a portion of the one or more nucleicacid templates.
 2. The method of claim 1, wherein the one more nucleicacid templates are RNA.
 3. The method of claim 2, wherein the nucleicacid templates are mRNA.
 4. The method of claim 1, wherein thesynthesized molecule is labeled.